RESEARCH ARTICLE 1

The second site modifier, Sympathy for the ligule, encodes a homolog of 2 Arabidopsis ENHANCED DISEASE RESISTANCE4 and rescues the 3 Liguleless narrow maize mutant 4

Alyssa Anderson1,2, Brian St. Aubin1,2, #, María Jazmín Abraham-Juárez2^, Samuel 5 Leiboff2, Zhouxin Shen3, Steve Briggs3, Jacob O. Brunkard2, Sarah Hake*2 6

1. Co-first authors7 2. Plant Gene Expression Center, USDA-ARS and University of California Berkeley, 8008 Buchanan St. Albany CA 94710 9 3. Division of Biological Sciences, University of California San Diego, La Jolla, CA 9209310 * corresponding author, hake@berkeley.edu11 # present address: Department of Plant Biology, Michigan State University, 612 Wilson Rd, East 12 Lansing 48824 13 ^ present address: Instituto Potosino de Investigación Científica y Tecnológica (IPICYT), San 14 Luis Potosí, Mexico. 15

16 Short title: Sympathy for the ligule: a maize homolog of AtEDR4 17 One-sentence summary: Allelic variation at Sympathy for the ligule affects growth and 18 development of Liguleless narrow maize mutants. 19

20 The author responsible for distribution of materials integral to the findings presented in this article 21 in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: 22 Sarah Hake (hake@berkeley.edu). 23

24 ABSTRACT 25 liguleless narrow1 encodes a plasma membrane-localized receptor-like kinase required for normal 26 development of Zea mays (maize) leaves, internodes, and inflorescences. The semi-dominant Lgn-27 R mutation lacks kinase activity and phenotypic severity is dependent on inbred background. We 28 created near isogenic lines and assayed the phenotype in multiple environments. Lgn-R plants that 29 carry the B73 version of Sympathy for the ligule (Sol-B) fail to grow under hot conditions but those 30 that carry the Mo17 version (Sol-M) survive at hot temperatures and are significantly taller at cool 31 temperatures. To identify Sol, we used recombinant mapping and analyzed the Lgn-R phenotype 32 in additional inbred backgrounds. We identified amino acid sequence variations in 33 GRMZM2G075262 that segregate with severity of the Lgn-R phenotypes. This gene is expressed 34 at high levels in Lgn-R B73 but expression drops to non-mutant levels with one copy of Sol-M. An 35 EMS mutation solidified the identity of SOL as a maize homolog of Arabidopsis thaliana 36 ENHANCED DISEASE RESISTANCE4 (EDR4). SOL, like EDR4, is induced in response to 37 pathogen-associated molecular patterns such as flg22. Integrated transcriptomic and 38 phosphoproteomic analyses suggest that Lgn-R plants constitutively activate an immune signaling 39 cascade that induces temperature-sensitive responses in addition to defects in leaf development. 40 We propose that aspects of the severe Lgn-R developmental phenotype result from constitutive 41 defense induction and that SOL potentially functions in repressing this response in Mo17 but not 42 B73. Identification of LGN and its interaction with SOL provides insight into the integration of 43 developmental control and immune responses. 44

Plant Cell Advance Publication. Published on June 19, 2019, doi:10.1105/tpc.18.00840

2

45

INTRODUCTION 46

The expressivity of a mutant phenotype is often dependent on other genes. These second site 47

modifiers have been identified through mutagenesis screens and crosses to different backgrounds. 48

Background dependent modifiers have been found for developmental pathways as diverse as 49

tomato (Solanum lycopersicum) fruit size and Drosophila bristle number (1, 2). Maize (Zea mays) 50

is particularly rich for identification of modifiers because of the high genetic variation among 51

maize inbred lines: to illustrate this point, the coding sequence variation between two maize 52

inbreds is as great as the coding sequence variation between humans and chimpanzees (3), and the 53

noncoding intergenic space is also remarkably variable between inbreds (4). Examples of 54

modifiers in maize include those for seed protein content (5), lesion mimic expressivity (6), and 55

virescence (7). Few modifiers that affect pleiotropic phenotypes, however, have been analyzed at 56

the molecular level. 57

Liguleless narrow-R (R for reference allele) has striking developmental phenotypes caused by an 58

EMS-induced point mutation that eliminates protein kinase activity (8). As a heterozygote in the 59

inbred line B73, Lgn-R plants are short with narrow leaves and reduced inflorescences. The adult 60

leaves fail to properly develop ligules and auricles, structures that are located at the blade/sheath 61

boundary in grasses. The ligule keeps water and debris from entering into the stem where the 62

axillary bud forms (9). The auricle allows the blade to tilt back and maximize exposure to sunlight. 63

The expressivity of the Lgn-R phenotype is background-dependent. While the phenotype is severe 64

in B73, plant height, leaf width, and inflorescence and ligule development are all restored to near 65

wild-type in Mo17 (10). To investigate these background differences, we crossed Lgn-R in B73 to 66

the intermated Mo17xB73 recombinant inbred lines (IBM lines) (11) and conducted a QTL 67

analysis. Sympathy for the ligule (Sol) was identified as a main effect QTL on chromosome 1 (10). 68

A genotype-by-environment (GxE) effect was discovered by growing the population in two 69

different environments. Lgn-R B73 mutants failed to grow in the hot summers of West Lafayette, 70

IN but survived to reproduce in the cooler weather of Albany, CA. Phenotypic expressivity that is 71

3

dependent on background and temperature is often seen in autoimmune mutants in other plant 72

species (12). 73

Here, we describe Lgn-R in the near isogenic line (NIL) that contains Mo17 at Sol in a 74

predominantly B73 background and identify the gene for Sol by fine-mapping and expression 75

analysis. Sol encodes a homolog of Arabidopsis ENHANCED DISEASE RESISTANCE4 (EDR4). 76

EDR4 binds to ENHANCED DISEASE RESISTANCE1 (EDR1), a MAP kinase kinase kinase) 77

and localizes it to the site of hyphal penetration pegs (13). Like EDR4, Sol is transcriptionally 78

expressed in response to treatment with pathogen associated molecule patterns (PAMPs). Sol 79

transcript levels are also increased in expression under normal growth conditions in Lgn-R B73 80

but not in Lgn-R NIL and non-mutant B73 or Mo17 siblings. Thus, the NIL has both developmental 81

phenotypes and Sol expression levels that more closely resemble its non-mutant siblings. Based 82

on transcriptomic, proteomic, and phenotypic analyses, we hypothesize that the Lgn-R mutation 83

triggers an autoimmune syndrome and that the NIL modifies this response. Our data begin to 84

highlight hypotheses to explain the modifying behavior of Sol on Lgn-R and identify important 85

molecular components that contribute to the Lgn-R phenotype. 86

87

RESULTS 88

The Near Isogenic Line dampens the Lgn-R mutant phenotype 89

To investigate the difference in expressivity between B73 and Mo17, we created near isogenic 90

lines (NILs) by first crossing Lgn-R to the IBM recombinant inbreds (10) and then back-crossing 91

the least severe Lgn-R individuals to B73 for a minimum of four generations. We refer to the Mo17 92

Sol locus as Sol-M and the B73 locus as Sol-B. Phenotypes were assessed in Lgn-R heterozygotes, 93

and plants were either Sol-M/Sol-B (NIL) or Sol-B/Sol-B (B73). 94

We grew Lgn-R NIL plants over multiple seasons to determine the effect on plant architecture in 95

comparison with Lgn-R in the B73 and Mo17 backgrounds (Figure 1A-C). Whereas Lgn-R Mo17 96

has nearly the same height and leaf width as the non-mutant Mo17 inbred, both height and width 97

are reduced in the NIL, and most severe in B73 (Figure 1D-G; Supplemental Figure 1A, B). In 98

4

Albany, CA, non-mutant plants averaged 179.4 cm (±10.5) in height and 10 cm (±0.6) in leaf 99

width, Lgn-R NIL plants averaged 147.4 cm (±10.2) in height and 7.5 cm (±0.9) in leaf width, and 100

Lgn-R B73 averaged only 102.4 cm (±22.4) for plant height and 4.1 cm (±0.7) for leaf width. Ear 101

development was also restored in Lgn-R NIL plants compared to Lgn-R B73 (Figure 1A, H). 102

Non-mutant B73 and Mo17 plants as well as Lgn-R Mo17 mutants had morphologically normal 103

ligules and auricles located between distinct blade and sheath regions. The ligule in Lgn-R NIL 104

plants formed across the leaf but the auricle was only partially restored and, as previously described 105

(8), Lgn-R B73 plants never developed a complete ligule or auricle in adult leaves (Figure 1B, C). 106

To elucidate the dosage effect of Sol-M on Lgn-R in the B73 background, we self-pollinated the 107

Lgn-R NIL and analyzed the segregating genotypes in the greenhouse. Of the Lgn-R heterozygotes, 108

the NIL and Sol-M/Sol-M plants had greater plant heights and leaf widths than their B73 siblings 109

(height, width: p < 2e-4, p < 2e-6) but there were no significant differences between the NIL and 110

Sol-M/Sol-M individuals (Figure 1I, J; Supplemental Figure 1C). The ligule itself however, was 111

near normal with two copies of Sol-M (Figure 1K). Among the Lgn-R homozygotes, the Sol-M 112

allele exhibited a clear dosage effect in that the NIL had statistically greater heights and widths 113

than Sol-B/Sol-B individuals (p < 0.007, p < 0.004) but had significantly smaller heights and widths 114

than Sol-M/Sol-M plants (p < 0.02, p < 0e-00) (Figure 1I, J; Supplemental Figure 1C). Thus, one 115

copy of Sol-M can improve growth of both Lgn-R heterozygotes and homozygotes, but Lgn-R 116

homozygotes retain multiple defects. 117

118

Sol rescues temperature-dependent Lgn-R lethality 119

A genotype by environment (GxE) effect was observed when the original Lgn-R x IBM lines were 120

analyzed in West Lafayette, IN and Albany, CA (10). To explore the underlying cause, plants were 121

grown at different temperatures. Lgn-R NIL plants segregating for Sol-M were grown in Davis, 122

CA, where average daytime temperatures during our field seasons were 33.1°C compared to that 123

of West Lafayette, IN at 29.0°C and Albany, CA at 23.2°C (data available at 124

http://www.ncdc.noaa.gov). Nights have similar temperatures in Davis and Albany and are 125

slightly warmer in West Lafayette. 126

5

Lgn-R plants showed significant GxE effects between the Albany, CA and Davis, CA fields 127

(Figure 1D-G). A one-way ANOVA with a Tukey Posthoc test (95% CI) found significant effects 128

from genotype (p = 2e-16), location (p = 2e-16), and genotype by location interaction (p = 1.4e-129

11) when comparing the Lgn-R B73 and Lgn-R Mo17 populations as well as significant effects 130

from genotype (p = 2.7e-11), location (p = 0.0019), and genotype by location interaction (p = 4e-131

7) when comparing Lgn-R NIL to Lgn-R B73 siblings. All pairwise comparisons can be found in 132

Supplemental Figure 1A, B. For example, during the 2015 Davis field season, 17 out of 19 Lgn-R 133

plants that were homozygous B73 at the Sol locus died within five weeks post-germination (Figure 134

2A, B). By contrast, only 1 out of 10 Lgn-R NIL plants died. None of the Lgn-R plants died in 135

Albany. The Lgn-R NIL plants that survived in Davis were shorter and had narrower leaves 136

compared to their Albany counterparts. Non-mutant plants survived at both locations, and were, 137

in fact, taller in Davis (Figure 2B). 138

To determine whether growth at high temperature is sufficient to trigger Lgn-R lethality, we grew 139

segregating Lgn-R NIL families under two different temperature regimes in otherwise constant 140

growth chambers (15-32°C and 11-21°C). The cooler temperature within these ranges was 141

maintained at night and the warmer temperature was used during the day in an attempt to mimic 142

field conditions. In the cool cycling growth chamber, the genotypes had nearly identical 143

phenotypes whereas Lgn-R NIL plants were noticeably more severe in the warm cycling chamber 144

and Lgn-R B73 plants were the most strongly affected (Figure 2C). After removal from the warm 145

cycling chamber, non-mutant and Lgn-R NIL plants continued to grow and form reproductive 146

tissues while Lgn-R B73 plants never recovered (Figure 2D). We also grew the segregating 147

populations at either a constant 17°C or 30°C. Significant reduction in plant height and leaf width 148

was seen across all genotypes at 30°C compared to 17°C with Lgn-R B73 the most strongly 149

affected by the 30°C condition. Intriguingly, some of the phenotypic differences seen under normal 150

field conditions were eliminated in the 17°C growth chamber experiment. Although leaves are still 151

narrower in Lgn-R B73 compared to NIL and non-mutant siblings, plant height was not 152

significantly different between any of the genotypes at this temperature and ligule development 153

was restored in Lgn-R B73 plants at 17°C (Figure 2E-H, Supplemental Figure 1D). In summary, 154

Lgn-R phenotypes are more severe at higher temperatures and rescued at cooler temperatures and 155

in the presence of the Mo17 allele of Sol. 156

6

Sol is an orthologue of ENHANCED DISEASE RESISTANCE 4 157

To identify the Sol modifier, we genotyped plants from Lgn-R NIL families and scored the 158

phenotypes (Supplemental Table 1, Materials and Methods). With recombinant mapping 159

techniques we were able to place Sol between IDP1489 at 56,572K bp and bnlg2238 at 55,080K 160

bp on chromosome 1 (Figure 3A). Based on the idea that syntenic genes are most likely to be 161

expressed as proteins (14), we used the Comparative Genomics Website (CoGe) to identify four 162

maize genes in the interval that have orthologs in sorghum, rice, Setaria and Brachypodium (15). 163

The syntenic genes included GRMZM2G075262, a gene of unknown function, 164

GRMZM2G072892, a putative LUNG-7 transmembrane receptor, GRMZM2G049211, a putative 165

vacuolar sorting receptor precursor, and GRMZM2G119850, a putative receptor-like kinase. 166

To continue investigating this interval, we crossed Lgn-R in B73 to the NAM Founder lines (16) 167

to determine which additional lines rescued the Lgn-R mutation at the Sol locus. 20 F1 Lgn-R 168

hybrid populations were grown in Indiana, where Lgn-R in B73 is severe. One Lgn-R hybrid, 169

Ms71/B73, showed a lethal phenotype. All other hybrids rescued the Lgn-R phenotype to some 170

extent (Figure 3B), though the rescued phenotype is not necessarily due to the Sol locus. Eight of 171

the hybrids were successively back-crossed a minimum of three generations to B73 using the most 172

rescued Lgn-R plant in each generation. Given the severe phenotype of Lgn-R in the Ms71/B73 173

hybrid, we also crossed Lgn-R in the Mo17 background to Ms71 and back-crossed to Ms71 at least 174

two generations prior to analysis. To assess environment and background effects, we planted these 175

segregating families in Davis and Albany during three field seasons. We determined plant height, 176

leaf width and the genotype of Sol for each family. We determined that a line rescued Lgn-R if it 177

displayed increased plant height and leaf width when heterozygous at the Sol locus compared to 178

Sol-B/Sol-B or Sol-Ms71/Sol-Ms71 siblings. 179

Of the NAM founder lines examined, four did not rescue Lgn-R at Sol (Ms71, CML228, CML247, 180

and Nc358) and three lines displayed rescued phenotypes (Nc350, Tzi8, and M162W) (Figure 3C-181

I, Supplemental Figure 1E-K). Of the severe lines, Lgn-R in the Ms71 inbred behaved similarly 182

to Lgn-R B73. The average plant height and leaf width measurements for Lgn-R Sol-Ms71/Sol-183

Ms71 in Albany were 144.2 cm (±35.7) and 5.5 cm (±1.1) and in Davis they averaged 40.7 cm 184

(±43.5) and 3.2 cm (±1.8) compared to the Lgn-R Sol-M/Sol-Ms71 measurements of 156 cm (± 185

7

30.6) and 8.9 cm (± 0.7) in Albany and 112.4 cm (± 26.4) and 5.1 cm (± 1.3) in Davis (Figure 3C). 186

A one-way ANOVA followed by a Tukey Posthoc test (95% CI) found significant effects from 187

genotype (p = 8.8e-7), location (p = 0.0004), and genotype by location interaction (p = 0.007) for 188

the family. The significance for individual comparisons can be found in Supplemental Figure 1E. 189

CML228 was also not capable of rescuing Lgn-R at Sol. Sol-CML228/Sol-B plants have heights 190

and widths that are not statistically different from Sol-B/Sol-B relatives at either field, though 191

significant GxE effects were still observed for individuals within a given genotype (Figure 3D; 192

Supplemental Figure 1F). This same relationship holds true for the inbred lines CML247 and 193

NC358 (Figure 3E, F; Supplemental Figure 1G, H). 194

In contrast to the severe lines, three additional lines rescued the Lgn-R phenotype when 195

heterozygous at Sol. Average plant heights and leaf widths for the Sol-Nc350/Sol-B heterozygotes 196

were, respectively, in Albany 141.6 cm (±14.22) and 6.7 cm (±0.7) and in Davis 72.0 (±23.5) and 197

3.5 cm (±1.0). These averages are all greater than their Sol-B/Sol-B counterparts, which measured 198

116.4 (±13.4) and 5.3 cm (±0.7) in Albany and 52.5 (±14.7) and 1.75 cm (±.5) in Davis. A one-199

way ANOVA followed by a Tukey Posthoc test (95% CI) found significant effects from genotype 200

(p = 2e-16), location (p = 0.0002), and genotype by location interaction (p = 1.6e-11) for the family 201

(Figure 3G, Supplemental Figure 1I). These same measurements for Sol-Tzi8/Sol-B heterozygotes 202

were 158.5 cm (±15.4) and 6.5 cm (±0.4) in Albany and were significantly increased over their 203

Sol-B/Sol-B relatives which measured 120.4 cm (± 12.5) and 4.9 (± 0.4), with the same trends in 204

Davis. A one-way ANOVA followed by a Tukey Posthoc test (95% CI) found significant effects 205

from genotype (p = 1.1e-11), location (p = 6.4e-13), and genotype by location interaction (p = 206

5.0e-11) for the family (Figure 3H, Supplemental Figure 1J). The third rescuing line, M162W, 207

showed a similar pattern (Figure 3I, Supplemental Figure 1K). 208

Analysis of SNP data from Panzea (17) for the genes in the Sol mapping interval revealed coding 209

sequence variation in GRMZM2G075262 that suggests this gene is responsible for rescuing Lgn-210

R phenotypic defects. Of the four genes, the GRMZM2G049211 alleles were identical, 211

GRMZM2G072892 had a single synonymous SNP, GRMZM2G119850 had 4 nonsynonymous 212

SNPs and 18 synonymous SNPs, and GRMZM2G075262 had the most differences, with 12 213

nonsynonymous SNPs, 26 synonymous SNPs, and 8 indels. The indels were discovered upon 214

direct sequencing of the coding region of this gene in Mo17, B73 and the inbred lines used in 215

8

crosses. We then asked whether sequence variation correlated with a line’s ability to rescue Lgn-216

R. We found no informative correlations based on the SNPs in GRMZM2G119850. By contrast, 217

6 of the 12 nonsynonymous SNPs in the GRMZM2G075262 alleles correlated with the ability of 218

Sol to rescue Lgn-R and 4 of the 7 Indels correlated with rescuing ability. Indeed, lines that could 219

rescue Lgn-R had the Mo17 version of these SNPs and indels, while the lines that could not rescue 220

had the B73 versions (Figure 4A, Supplemental Figure 2A). CML228 and CML247 are exceptions 221

to this pattern: they have Mo17-like sequences but do not rescue Lgn-R when heterozygous with 222

B73 at Sol. 223

We investigated the expression of the four genes from the Sol QTL interval via RT-qPCR. This 224

analysis revealed that GRMZM2G075262 transcript levels are significantly higher in Lgn-R B73 225

shoot apices than in Lgn-R NIL and non-mutant siblings. None of the other syntenic genes in the 226

interval had trends in expression levels that directly correlated with severity of the Lgn-R 227

phenotype (Figure 4B). 228

To investigate why CML228 and CML247 have GRMZM2G075262 sequences similar to Mo17 229

but fail to rescue, we carried out RT-qPCR in populations segregating for Lgn-R in these families. 230

For comparison, we used Ms71, which has the B73 Sol haplotype and results in a severe Lgn-R 231

phenotype, and Nc350, which has the Mo17 Sol haplotype and rescues Lgn-R (Figure 4A). As 232

hypothesized, Sol is induced in Lgn-R plants in the Ms71 background, but not in Lgn-R plants in 233

a Sol-Nc350/Sol-B background (Figure 4C). We found that Sol transcripts are increased in lines 234

that are Lgn-R and CML228/Sol-B. Results with CML247 were inconsistent across replicates. 235

This result shows that Sol-Nc350, like Sol-M, is capable of reducing Sol-B expression in the 236

heterozygous condition, but that Sol-CML228 is not despite the similarity in sequence to Sol-M. 237

Thus, allelic differences in protein sequence and in regulation of gene expression may both 238

contribute to variation at Sol. 239

A mutagenesis screen supported our hypothesis that GRMZM2G075262 is the gene responsible 240

for the effects of the Sol locus on the Lgn-R phenotype. Lgn-R plants that had been backcrossed 241

three times to Mo17 were mutagenized with EMS and crossed onto B73. The resulting 2,000 242

kernels were planted in the field and scored for Lgn-R phenotypes. Whereas almost all of the 1,000 243

Lgn-R plants showed partial rescue of plant height and leaf width, we identified one plant with a 244

9

more severe Lgn-R phenotype that, when sequenced at GRMZM2G075262, contained a G489E 245

missense mutation in Sol-M (Figure 4A, blue line, Figure 4D, Supplemental Figure 2A blue box). 246

We did not observe any developmental phenotypes of Sol-G489E without Lgn-R in the 247

background. 248

GRMZM2G075262, henceforth referred to as Sol, encodes a homolog of A. thaliana ENHANCED 249

DISEASE RESISTANCE4 (EDR4). We used BLASTp to identify similar proteins in representative 250

plant species, including several grasses and eudicots, and constructed a phylogenetic tree to 251

describe the relationship of these proteins. SOL and AtEDR4 are in a monophyletic clade of 252

closely-related proteins, confirming that the two are homologs (Figure 4E). AtEDR4 and SOL 253

share 61.36% identity across 42% of the protein which includes a C-terminal Zn ribbon 12 domain 254

and an N-terminal Zn finger-like domain (Supplemental Figure 2B). The maize genome includes 255

another three EDR4-like genes within the same clade as Sol, however these have no assigned 256

function and have not been previously investigated. The only EDR4-like protein that has been 257

intensively studied is AtEDR4 (13). 258

We also determined the cellular compartment for SOL-M and SOL-B. Using Nicotiana 259

benthamiana for 35S:Sol-B-YFP and 35S:Sol-M-YFP transient expression, we determined that 260

SOL-B localizes to the cell periphery and nucleus. SOL-M, by contrast, localized only to the cell 261

periphery (Figure 5A-F). Although these experiments were carried out in N. benthamiana and not 262

maize, they support the idea that SOL-B and SOL-M have functional differences. Our combined 263

data from analysis of Lgn-R in multiple inbreds, gene expression levels in different genetic 264

backgrounds, a revertant screen, and cell localization all strongly support the identity of Sol as 265

GRMZM2G075262, a maize homolog of EDR4. 266

Sol expression is induced by elicitors of innate immunity 267

AtEDR4 expression shows induction one hour after treatment with the bacterial elicitors flg22 and 268

HrpZ (18). To test if Sol is also transcriptionally induced in response to pathogen elicitors, we 269

measured Sol transcript levels 10, 30, 60 minutes, and 24 hours after treatment with chitin (a fungal 270

elicitor) or water (control). Endochitinase PR4 (PR4), known to be induced in maize by chitin and 271

other elicitors (19), was used to confirm the efficacy of the treatments. By RT-qPCR, we found 272

10

that Sol was strongly increased in expression within one hour after treatment with chitin in B73 273

(wild-type) leaves (Figure 5G). We also treated B73 with flg22 (a bacterial elicitor), and treated 274

Lgn-R B73 tissue with chitin, flg22, or water and collected samples at the 60-minute timepoint. 275

We found that Sol in B73 was also induced by flg22 at the 60-minute time point, whereas Sol 276

levels were not statistically induced in Lgn-R after treatment with chitin or flg22 (Figure 5H) 277

presumably because mock-treated Lgn-R leaves already showed significant induction levels 278

compared to the non-mutant B73 controls. In fact, we observed a significant reduction in Sol 279

expression at the 60-minute time point in Lgn-R tissue treated with chitin compared to the control. 280

The induction of Sol by elicitors indicates conservation of this particular transcriptional response 281

across species, supporting its identity as a homolog of EDR4. 282

Lgn-R putatively triggers a MAPK innate immunity signaling cascade 283

To narrow our search for pathways affected by the Lgn-R mutation, we used a transcriptomic 284

approach to identify differentially expressed genes (DEGs) in Lgn-R versus non-mutant siblings. 285

We found 1,568 DEGs (FDR < 0.05) in Lgn-R shoot apices compared to non-mutant siblings, of 286

which 1,119 were induced and 448 were repressed. A large number of the Lgn-R DEGs are related 287

to disease resistance, including induction of genes that encode receptor-like kinases (RLKs), 288

WRKY transcription factors, several classical PATHOGENESIS-RELATED PROTEINS, and 289

nucleotide-binding leucine-rich repeat Nod-Like Receptors (NLRs) (Figure 6A-C, Supplemental 290

Data Set 1). This transcriptional signature suggests that an innate immunity signaling cascade is 291

activated in Lgn-R mutants in the absence of pathogen infection. Therefore, we hypothesize that 292

loss of LGN activity causes an “autoimmune syndrome” with developmental consequences. 293

To identify possible substrates of Lgn-R, we took a global phosphoproteomic approach. Three 294

biological replicates of shoot apex tissue were collected from Lgn-R B73 and non-mutant siblings. 295

We detected phosphopeptides in proteins encoded by 3,000 different maize genes across all 296

samples. 35 proteins were phosphorylated in all wild-type samples, but not in any Lgn-R samples 297

(Supplemental Table 2); these could therefore be peptides that are uniquely phosphorylated by 298

LGN or downstream of LGN signaling. There is no clear pattern among these potential substrates, 299

although these proteins include a number of transcription factors, chromatin remodeling factors, 300

11

proteins involved in cell wall synthesis, and cell cycle regulators that could impact Lgn-R 301

phenotypes. 302

EDR4 is proposed to suppress MAP kinase (MAPK)-mediated immune responses by controlling 303

the localization of a MAP kinase kinase kinase (MAP3K), called ENHANCED DISEASE 304

RESISTANCE1 (EDR1 or AtMAP3Kδ3), that acts as an upstream repressor of AtMPK3/AtMPK6 305

activity (13). Moreover, AtMPK3/AtMPK6-mediated defense signaling is suppressed at cool 306

temperatures (20), similar to the suppression of Lgn-R phenotypes at cool temperatures. These 307

correlations led us to speculate that Sol could function by interacting with an immunity-related 308

MAPK signaling cascade that is activated in the Lgn-R mutant. To investigate this possibility, we 309

searched for signs of a differentially-phosphorylated signaling cascade in the Lgn-R 310

phosphoproteome (compared to the wild-type phosphoproteome), focusing on phosphopeptides 311

identified in our dataset that have strong similarity to well-characterized phosphorylation sites on 312

proteins in other plant species. With this targeted approach, we identified a putative signaling 313

cascade that could act downstream of LGN-R to promote expression of pathogen defense-related 314

genes and disrupt development. Although not statistically significant, we did identify putative 315

differentially phosphorylated sites on homologues of BIR3, BAK1, CDG1, BSU1, SHAGGY, 316

MAP3K (YODA-like), MAP2Ks, MAPKs, and WRKYs that are consistent with activation of this 317

innate immunity MAP kinase signaling cascade in Lgn-R, but not in non-mutant siblings 318

(Supplemental Data Set 2). Alignments of the shared phosphosites in BAK1, BSU1, and 319

SHAGGY-Like BIN2 between maize and Arabidopsis are shown in Supplemental Figure 2C-E. 320

Another canonical MAPK substrate, MKP1, is also hyperphosphorylated in Lgn-R, further 321

indicating that LGN regulates MAPK signaling (21). Thus, we suggest that LGN could act as an 322

upstream repressor of a BAK1-SHAGGY-MAPK signaling network that induces expression of 323

pathogen defense genes. 324

325

DISCUSSION 326

Lgn-R is a semi-dominant maize mutant with striking leaf defects. The phenotype is severe in B73 327

and nearly wild type in Mo17. Analysis of a near isogenic line that segregates for Mo17 at the Sol 328

12

locus showed that Sol-M rescues severe aspects of the Lgn-R phenotype. One copy of Sol-M 329

partially restores the plant height, leaf width, ligule development, ear development, and provides 330

heat stress-survival of the Lgn-R mutant. We mapped Sol to a region containing four genes. 331

Subsequent SNP and expression analysis, as well as a mutagenesis screen, strongly support the 332

hypothesis that GRMZM2G075262, a maize homolog of ENHANCED DISEASE RESISTANCE4, 333

is responsible for the Sol phenotypes. Integrated transcriptomics and phosphoproteomics suggest 334

that the Lgn-R mutation triggers a pathogen defense transcriptional program in the absence of 335

pathogen infection. Sol-M, but not Sol-B, rescues many of the development defects and may act in 336

a feedback loop to suppress immune system activation. 337

Lgn-R resembles other mutants with constitutively activated pathogen defense responses in certain 338

regards. Auto-immune mutants are characterized by dwarfism, upregulation of biotic stress genes, 339

and sensitivity to growth conditions and genetic background (12). Plant growth is compromised, 340

presumably because more energy is allocated to defense than to growth and development (22). 341

Although few maize autoimmune mutants have been described (23), Lgn-R appears unique in its 342

developmental leaf defects such as the missing ligule and narrow leaves. Many autoimmune 343

mutants caused by lesions in NLR genes (or related pathways), including bonsai, chs3-1, chs2-1 344

and rpp4 in Arabidopsis and Rp1-D21 in maize, have severe phenotypes at low temperatures that 345

are rescued at higher temperatures (24-30). Lgn-R follows an opposite pattern in terms of 346

temperature expressivity because its phenotypes are most severe at high temperature and rescued 347

at lower temperatures. We hypothesize that this difference in temperature sensitivity is due to 348

activation of distinct pathogen defense signaling networks: NLR-mediated defenses (sometimes 349

called “Effector-Triggered Immunity”, or ETI) are commonly active at low temperatures and 350

suppressed at high temperatures, whereas MAPK-mediated defenses (sometimes called “Pattern-351

Triggered Immunity”, or PTI) are commonly active at high temperatures and suppressed at low 352

temperatures (20). The extreme temperature sensitivity and phosphoproteome of Lgn-R suggest it 353

is a mutation in PTI. The mechanisms by which temperature influences pathogenicity, NLR 354

activity, and MAPK signaling remain unclear (31, 32); future studies on the precise molecular 355

functions of LGN under different temperatures could help illuminate how plant defenses are 356

impacted by the environment in agricultural crops. 357

13

Lgn-R developmental defects are only partially rescued by the NIL; the ligule is still incomplete 358

and leaves are narrow compared to Lgn-R in Mo17. In the greenhouse, increasing the copy number 359

of Sol-M does not statistically change the height or leaf width of Lgn-R heterozygotes, but does 360

restore a normal ligule. Sol-M statistically improves growth of Lgn-R homozygotes in a dosage-361

dependent manner. One copy increases leaf width and plant height and two copies further improves 362

their growth, but they are still clearly mutants. These results demonstrate the importance of at least 363

one functional copy of LGN for complete rescue by Sol-M. 364

365

When Lgn-R was crossed into additional inbred lines, a pattern of amino acid substitutions and 366

indels in Sol became apparent between the rescuing and severe inbred lines. There were, however, 367

two exceptions to this pattern, CML247 and CML228. Further investigation showed that, despite 368

their Mo17-like coding regions, at least one, CML228, had significantly increased mRNA levels 369

in the Lgn-R background when heterozygous with Sol-B. Promoter differences could help to 370

explain this regulatory difference as well as the failure of these alleles to rescue Lgn-R. 371

In further support of the idea that relative expression levels of Sol could lead to functional 372

differences, EDR4 and Sol are both transiently induced 60 minutes after cells are exposed to 373

receptor like kinase (RLK) elicitors such as flg22. This result suggests that Sol may be a pathogen-374

response gene, and that SOL-M may act in a negative feedback loop to limit constitutive activation 375

of defense-related MAPK signaling cascades. EDR4 functions in repressing immune responses by 376

relocating EDR1, which has a repressive effect on MAPK signaling cascades, to the plasma 377

membrane in regions of hyphal penetration. The EDR4 and EDR1 interaction is dependent on the 378

presence of the first 150 amino acids in EDR4 (13). Interestingly, the majority of Indels and 379

nonsynonymous mutations between SOL-B and SOL-M are within the first 150 amino acids of the 380

protein. This finding implies that amino acid differences could also be at the heart of the functional 381

differences between the two versions. Additionally, EDR4 is localized at the membrane (13), as 382

are SOL-M and SOL-B when transiently expressed in Nicotiana benthamiana. Nuclear 383

localization of SOL-B and not SOL-M suggests either protein misregulation, sub-functionalization 384

of SOL-B, or simply heterologous misexpression of the protein. Nonetheless, our results suggest 385

a number of avenues for further investigation, all of which are potentially related. For example, 386

the amino acid differences could lead to different binding partners which in turn lead to alternate 387

localization and expression levels. Experiments to elucidate potential binding partners of SOL-M 388

14

versus SOL-B will help unveil functional differences between the two proteins. 389

We propose that LGN is capable of repressing immune responses through upstream 390

phosphorylation of a biotic defense signal transduction cascade (Figure 6D). LGN may also 391

function to promote leaf development through an independent signaling network. The ligule and 392

auricle defects found in Lgn-R are unique among auto-immune mutants and could be caused by a 393

distinct developmental pathway affected in the Lgn-R mutants. Recent studies have linked MAPK 394

signaling cascades to leaf angle phenotypes in rice (33). Perhaps LGN affects multiple MAPK 395

pathways, some leading to autoimmune responses and others leading to distinct developmental 396

events. Sol is transcriptionally downstream of the immune signaling cascade controlled by LGN 397

and becomes transcriptionally induced in the mutant background. We speculate that SOL-M has a 398

similar function to AtEDR4 and is capable of suppressing the immune response. SOL-B is 399

incapable of rescuing the Lgn-R phenotype, perhaps because it cannot suppress this immune 400

response. Although it is unclear how temperature influences these pathways, the observed 401

phenotypic patterns that we see are more closely associated with MAPK-based immune responses 402

than other types of immune signal transduction cascades (20). 403

The identification of the modifier Sol, with its significant genetic and molecular signatures among 404

the maize inbreds, could guide future breeding efforts to balance pathogen defense with overall 405

growth and yield. Further study of the developmental and immunity-related roles of LGN should 406

reveal new players in the realm of plant resource allocation when under pathogen attack and 407

unravel the complex signaling network underlying interactions between development and defense. 408

409

MATERIALS AND METHODS 410

Genetic material and crossing schemes: Lgn-R heterozygotes in a B73 background were crossed 411

to the IBM lines (11) and NAM founder lines (16) and scored in Indiana (10). The tallest Lgn-R 412

plants were back-crossed to B73 three to six times for mapping and evaluation. In earlier 413

generations the most rescued Lgn-R plant in each population was blindly used for the cross but 414

later generations used genotyping at Sol with the primers IDP1489 (Supplemental Table 1). 415

15

Because Lgn-R is very severe in an Ms71/B73 hybrid, Lgn-R in a B73 background was first crossed 416

to Mo17 two times and then crossed to Ms71 a minimum of two times as well. 417

EMS mutagenesis was carried out on pollen from Lgn-R heterozygotes backcrossed to 418

Mo17 three times and used to pollinate B73 ears. Approximately 2000 kernels were planted in the 419

field and Lgn-R plants were observed. Severe Lgn-R plants were crossed to B73 and non-mutants 420

were self-pollinated to create lines that segregated for Sol-M. Individuals that were homozygous 421

for Sol-M were sequenced using GRMZM2G075262 primers in Supplemental Table 1. 422

423

Field and phenotypic measurements: Three weeks after planting, plants were numbered and 424

tissue collected for DNA. Plants were scored at 5 weeks for a phenotypic assessment. Plant height 425

was taken at maturity. Leaf width was measured at the mid-point of the blade on the leaf above 426

the ear node. This leaf was consistently the longest and widest leaf for the non-mutants and usually 427

leaf 6 counting down from the top of the plant. When Lgn-R plants did not make ears, the leaf 428

measured was the 6th down from the top. In Davis, many of the Lgn-R plants died prior to maturity. 429

For one season, Lgn-R B73 plants were measured for plant height before they died. The average 430

high temperature for the months of June through August in Davis, CA is 33.1°C and 23.2°C in 431

Albany, CA (data from http://www.ncdc.noaa.gov). The GPS coordinates for the Albany and 432

Davis, CA fields are, respectively, (37.887, -122.300) and (38.535, -121.771). Oneway ANOVAs 433

and Tukey Posthoc tests were conducted in R. In a number of families, the amount of dead plants 434

in Davis did not allow for adequate statistical analysis via ANOVA. In these instances, Z statistics 435

were calculated according to the formula: 436

𝑧𝑧 = (𝑝𝑝1 − 𝑝𝑝2) �𝑝𝑝′ ∗ (1 − 𝑝𝑝′) ∗ �1𝑛𝑛1

+1𝑛𝑛2��� 437

Where p1 and p2 are the proportions for each genotype (i.e. dead plants in genotype/total plants in 438

genotype), p’ is the combined proportion (i.e. all dead plants/total plants analyzed) and n1, n2 are 439

the number of individuals analyzed /genotype. 440

441

Growth chamber and greenhouse conditions: Plants were grown in 16 hours full spectrum light 442

500 μm/m/s, 8 hours dark. In an attempt to mimic field condition temperatures, treatments included 443

the intervals of 15°C -32°C and 11°C -21°C, where high and low temperatures for each condition 444

were matched to light and dark respectively. A further experiment maintained the plants at a 445

16

constant 17°C or a constant 30°C. Watering was maintained sufficiently to maintain soil moisture, 446

and up to 1 cm standing in each tray immediately after watering. Soil humidity probes were used 447

to verify consistent hydration between temperature conditions. 448

The greenhouse conditions vary depending on weather but typically light intensity is 449

215μm/m/s, temperature ranges from 68-78◦F, humidity from 44-76%, and watering through drip 450

emitters occurs at a rate of 850mL/day. 451

452

Fine-mapping, markers and numbers of recombinants: To fine-map Sol, Lgn-R NIL plants 453

were back-crossed to B73 and progeny grown in Berkeley. All plants were measured for leaf width 454

and plant height. In 2012, markers phi109275 at 51,895,132 and bnlg1811 at 70,769,955 were used 455

to find 41 recombinants from a total of 627 plants. In 2013 and 2014 all plants were genotyped 456

regardless of ligule phenotype. Non-mutant plants that were recombinant at Sol were crossed with 457

Lgn-R B73 to assess the phenotype in the next generation. Recombinants that were Lgn-R were 458

crossed to B73 to confirm the phenotype in the next generation. Additional markers were used to 459

refine the position of recombinants to the region between the markers bnlg2238 and IDP1489. 460

Primer sequences are in Supplemental Table 1. 461

462

Inbred Sequence Analysis: The original SNP dataset used to analyze the four syntenic genes in 463

the Sol QTL interval was the Maize HapMapV2 data (34) found at Panzea.org (17). Using the 464

Genotype Search Tool, SNPs were identified from all relevant inbred lines in the interval on 465

chromosome 1 spanning the coding region of all interval genes. B73 RefGen_v3 mapping 466

locations were used. SNPs present in intronic sequences were ignored. The coding region of 467

GRMZM2G07562 was also directly sequenced in all studied inbred lines using primers described 468

in Supplemental Table 1. 469

470

Phylogenetic analysis: Sequences were curated via a BLASTp search against the non-redundant 471

protein sequence collection using the maize SOL sequence. All non-redundant matches with an e 472

value less than or equal to 0.001 and a minimum of 15% coverage were selected, downloaded and 473

aligned using MUSCLE (35). Sequences were manually curated to remove redundant sequences 474

submitted with non-redundant names and any obvious protein fragments that were less than 100 475

amino acids in length. This alignment (Supplemental Data set 3) was fed into MEGAX (36), which 476

17

we used to compute a Maximum Likelihood Tree with 75 bootstrapping replicates. A Jones-477

Taylor-Thornton model and Nearest Neighbor Interchange ML Heuristic were used while 478

assuming uniform evolutionary rates between sites. 479

A specialized function of the BLASTp suite was used to calculate % identity for a SOL, AtEDR4 480

alignment. Both sequences were entered individually using the two sequence option in BLASTp. 481

These sequences were then directly aligned and compared using the BLASTp software (37). 482

483

PAMP treatment and RT-qPCR analysis: Leaf 4 tissue of 4-week-old plants was collected and 484

PAMP or mock treatments were applied according to the method previously described (19). The 485

PAMPs used include flagellin 22 (GenScript) and Hexa-N-acetylchitohexaose (Cayman Chemical 486

Company). Leaf tissue was taken from at least three separate plants per genotype per treatment per 487

timepoint. Total RNA was extracted from these tissues using Trizol Reagent (Invitrogen) 488

according to the manufacturer’s specifications and cDNA was generated using the ThermoFisher 489

Superscript III Kits with OligoDt following the manufacturers recommendations. Two step RT-490

qPCR (98°C 0.2s, 55°C 0.5s) was conducted on a BioRad CFX96 Real-Time System using primers 491

for the housekeeping gene Gapdh to normalize the expression of our target genes: Sol and Pr4. All 492

primers are listed in Supplemental Table 1. Expression levels of the analyzed genes were 493

calculated according to the equation E = Peff (−ΔCt), where Peff is the primer set efficiency 494

calculated using LinRegPCR (38) and ΔCt was calculated by subtracting the cycle threshold (Ct) 495

value of the housekeeping gene from the Ct values of the gene analyzed. Fold changes were 496

calculated between the ratios of the expression levels of PAMP-treated and mock samples, and 497

expression levels were calculated for three biological replicates. 498

499

RNAseq material: Seeds of Lgn-R/IBM x B73 (BC5) were sown in large pots in the greenhouse 500

and 120 plants genotyped for Sol. Plants that were obviously Lgn-R were noted and others were 501

genotyped for Lgn-R resulting in 23 +/+ Sol-B/Sol-B, 15 +/+ Sol-M/Sol-B, 24 Lgn-R/+ Sol-B/Sol-502

B, 24 Lgn-R/+ Sol-M/Sol-B. Shoot apex tissue was harvested 4 weeks after planting. To be 503

consistent between genotypes, we made 2 biological replicates with 7 samples from each genotype. 504

ScriptSeq v2 RNA-Seq library preparation kit was used in accordance with the manufacture’s 505

guidelines for sequencing with illumina Hiseq 2000 as SR50. We assessed raw read quality and 506

checked for adapter contamination using fastqc507

18

(http://www.bioinformatics.babraham.ac.uk/projects/fastqc) and multiqc (39). Filtered reads were 508

aligned to the maize B73 genome AGPv3.30 using HISAT2 (40), and assigned to AGPv3.30 gene 509

models using HTSeq-counts (41), using Cyverse Atmosphere cyberinfrastructure (42). Counted 510

reads were tested for differential expression with edgeR using a GLM approach to account for 511

batch effects between repeated experiments and an FDR significance threshold of 0.05 (43) and 512

differentially-expressed fold changes used for additional plots. Gene functional categories were 513

assessed by a parametric test of gene enrichment (PAGE) with AgriGOv2 (44). For the RNAseq 514

analysis, we combined the two Lgn-R datasets (Sol-B/Sol-M and Sol-B/Sol-B) and combined the 515

two non-mutant datasets to increase the number of replicates in the analysis. 516

517

Phosphoproteome: Each pool had approximately 9 grams of shoot tissue isolated from three-518

week-old plants segregating for Lgn-R in the B73 background. Two grams of frozen tissue were 519

ground in liquid nitrogen by a mortar/pestle for 15 minutes to fine powder then transferred to a 520

50ml conical tube. Proteins were precipitated and washed by 50 ml -20°C acetone three times, 521

then by 50 ml -20°C methanol with 0.2mM Na3VO4 three times. Protein pellets were aliquoted 522

into four 2ml Eppendorf tubes per sample and dried in a SpeedVac at 4°C. 523

Ground tissue powders were suspended in extraction buffer (8M Urea/100mM Tris/5mM 524

Tris(2- carboxyethyl)phosphine (TCEP)/phosphatase inhibitors, pH 7). Proteins were precipitated 525

by adding 4 volumes of cold acetone and incubated at 4°C for 2 hours. Samples were centrifuged 526

at 4,000xg, 4°C for 5 minutes. Supernatant was removed and discarded. Proteins were re-527

suspended in Urea extraction buffer and precipitated by cold acetone. Protein pellets were washed 528

by cold methanol with 0.2mM Na3VO4 to further remove non-protein contaminants. Protein 529

pellets were suspended in extraction buffer (8M Urea/100mM Tris/5mM TCEP/phosphatase 530

inhibitors, pH 7). Proteins were first digested with Lys-C (Wako Chemicals, 125-05061) at 37°C 531

for 15 minutes. Protein solution was diluted 8 times to 1M urea with 100mM Tris and digested 532

with trypsin (Roche, 03708969001) for 4 hours. Reduced cysteines were alkylated by adding 533

10mM iodoacetamide and incubating at 37°C in the dark for 30 minutes. 534

Phosphopeptide enrichment was performed using CeO2 affinity capture. 1% colloidal 535

CeO2 (Sigma, 289744) was added to the acidified peptide solution (3% TFA, CeO2:peptide w:w 536

ratio = 1:10). After brief vortexing, CeO2 with captured phosphopeptides was spun down at 1,000g 537

for 1 minute. Supernatant was removed and the CeO2 pellet was washed with 1ml of 1% TFA. 538

19

Phosphopeptides were eluted by adding eluting buffer (200mM (NH4)2HPO4, 2M NH3.H2O, 539

10mM EDTA, pH 9.5, same volume as the added 1% colloidal CeO2) and vortexing briefly. CeO2 540

was precipitated by adding 10% formic acid with 100mM citric acid (same volume as the added 541

1% colloidal CeO2) to a final pH of 3. Samples were centrifuged at 16,100 g for 1 minute. 542

Supernatant containing phosphopeptides was removed for mass spectrometry analysis. 543

An Agilent 1100 HPLC system was used to deliver a flow rate of 600 nL min-1 to a custom 544

3-phase capillary chromatography column through a splitter. Column phases are a 30 cm long 545

reverse phase (RP1, 5μm Zorbax SB-C18, Agilent), 5 cm long strong cation exchange (SCX, 5μm 546

PolySulfoethyl, PolyLC), and 20 cm long reverse phase 2 (RP2, 2.5 μm BEH C18, Waters), with 547

the electrospray tip of the fused silica tubing pulled to a sharp tip (inner diameter <1 μm). Peptide 548

mixtures were loaded onto RP1, and the 3 sections were joined and mounted on a custom 549

electrospray adapter for on-line nested elutions. Peptides were eluted from RP1 section to SCX 550

section using a 0 to 80% acetonitrile gradient for 60 minutes, and then were fractionated by the 551

SCX column section using a series of 18 step salt gradients of ammonium acetate over 20 min, 552

followed by high-resolution reverse phase separation on the RP2 section of the column using an 553

acetonitrile gradient of 0 to 80% for 120 minutes. One 2D-nanoLC-MS/MS required 46 hours. 554

Spectra were acquired on LTQ Velos linear ion trap tandem mass spectrometers (Thermo 555

Electron Corporation, San Jose, CA) employing automated, data dependent acquisition. The mass 556

spectrometer was operated in positive ion mode with a source temperature of 325 °C and a spray 557

voltage of 3,000V. The maximum ion injection time is 50ms for MS and 100ms for MS/MS. As 558

a final fractionation step, gas phase separation in the ion trap was employed to separate the peptides 559

into 3 mass classes prior to scanning; the full MS scan range was divided into 3 smaller scan ranges 560

(400–750, 750–1,000, and 1,000–1,800 Da) to improve dynamic range. Each MS scan was 561

followed by 5 MS/MS scans of the most intense ions from the parent MS scan. A dynamic 562

exclusion of 1 minute was used to improve the duty cycle. 563

Raw data were extracted and searched using Spectrum Mill vB.06 (Agilent 564

Technologies). MS/MS spectra with a sequence tag length of 1 or less were considered to be poor 565

spectra and were discarded. The remaining MS/MS spectra were searched against maize B73 566

RefGen_v2 5b Filtered Gene Set downloaded from www.maizesequence.org. The enzyme 567

parameter was limited to full tryptic peptides with a maximum mis-cleavage of 1. All other search 568

20

parameters were set to SpectrumMill’s default settings (carbamidomethylation of cysteines, +/- 569

2.5 Da for precursor ions, +/- 0.7 Da for fragment ions, and a minimum matched peak intensity of 570

50%). Ox-Met, n-term pyro-Gln, and phosphorylation on Serine, Threonine, or Tyrosine were 571

defined as variable modifications for phosphoproteome data. A maximum of 2 modifications per 572

peptide was used. A 1:1 concatenated forward-reverse database was constructed to calculate the 573

false discovery rate (FDR). The tryptic peptides in the reverse database were compared to the 574

forward database, and were shuffled if they matched to any tryptic peptides from the forward 575

database. Common contaminants such as trypsin and keratin were included in the protein database. 576

There are 127,108 protein sequences in the protein database. Peptide cutoff scores were 577

dynamically assigned to each dataset to maintain the false discovery rate (FDR) less than 0.1% at 578

the peptide level. Phosphorylation sites were localized to a particular amino acid within a peptide 579

using the variable modification localization (VML) score in Agilent’s Spectrum Mill software. 580

Proteins that share common peptides were grouped using principles of parsimony to address the 581

protein database redundancy issue. Thus, proteins within the same group shared the same set or 582

subset of unique peptides. Protein abundance and phosphorylation levels were quantified via 583

spectral counting. Mass spectrometry runs (replicates) were normalized so that the total number 584

of spectral counts was equal for each run. Spectral counts from technical replicates, when present, 585

were then averaged to get the spectral counts for each biological replicate at the protein level. 586

587

Transient expression in N. benthamiana: To create 35S:Sol-B-YFP and 35S:Sol-M-YFP, the full-588

length Sol coding sequence was amplified from cDNA of B73 and Mo17 shoots with specific 589

primers (Supplemental Table 1), cloned into pENTR D and then recombined into pEarleyGate 101 590

(45) using the Gateway technology, according to the manufacturer’s instructions (Invitrogen). The 591

construct was transformed into Agrobacterium tumefaciens strain GV3101. Transient 592

transformation of Nicotiana benthamiana was performed as described (46). Forty-eight hours after 593

agroinfiltration, leaves were observed under LSM710 confocal microscopy (Zeiss) with 470 nm 594

excitation and 535 nm emission filters for YFP and 360 nm excitation and 488 nm emission filters 595

for DAPI signal. This experiment was repeated three times. 596

Accession Numbers 597

Sequence data from this work can be found under the following accession numbers: 598

21

Lgn: GRMZM2G134382, Sol: GRMZM2G075262, AtEDR4: AT5G05190, EcPR4:, 599 GRMZM2G129189, AtEDR1: AT1G08720, ZmBAK1-like: GRMZM2G115420, ZmBSU1-like: 600 GRMZM2G028700, ZmBIN2-like: GRMZM2G043350, ZmMKP1-like: GRMZM2G005350; 601 RNAseq data: Bioproject ID: PRJNA54785. 602

603 Supplemental Data 604

Supplemental Figure 1. 95% confidence intervals for all Tukey Posthoc tests, statistical support 605

for Figures 1-3. 606

Supplemental Figure 2. Expanded alignments of Maize and Arabidopsis proteins, supporting 607

Figures 4 and 6. 608

Supplemental Table 1. Primers used in study. 609

Supplemental Table 2. Phosphopeptides in B73 but not in Lgn-R. 610

Supplemental Data Set 1. Differentially expressed genes from RNAseq. 611

Supplemental Data Set 2. Phosphopeptides supporting a Map kinase signaling cascade in Lgn-R. 612

Supplemental Data Set 3. Text file of the alignment used for the phylogenetic analysis shown in 613

Figure 4E. 614

615

ACKNOWLEDGEMENTS 616

We thank the many students who helped in our summer field seasons including Yadanar Htike, 617

Sarah Vernallis, Melea Emunah, Nick Stivers, Ashley Noriega, Jamie Jeffries, Haley Kodak, 618

Alicia Ljungdahl. Thanks to Brianna Haining for sequencing EMS mutations, De Woods at the 619

USDA microscopy facility, greenhouse staff, UC Davis and UC Berkeley field staff. AA, MA-J 620

were supported by NSF IOS-1238202, SH by CRIS 5335-21000-013-00D, SL by NSF IOS-621

1612268, JB by NIH DP5OD023072, SB and ZC by NSF IOS 1546899. 622

AUTHOR CONTRIBUTIONS 623

SH and AA, designed the research and wrote the manuscript. AA and BS-A executed the 624

experiments, MJA-J did the localization in Nicotiana, ZS and SB carried out the 625

phosphoproteome analysis, JB and SL helped analyze the data. 626

627

22

FIGURE LEGENDS 628

Figure 1. The Sol-M modifier rescues the Lgn-R phenotype. 629

(A) Mature plant phenotypes pictured from left to right: Mo17, B73, Lgn-R Mo17, Lgn-R NIL, 630

Lgn-R B73. Ears are circled in red. (B) Adaxial view of ligules from leaf six. Lgn-R B73 ligule in 631

red box. (C) Abaxial view of ligules from leaf six in same order as (B). Auricles marked by red 632

angles. (D-G) Plant height and leaf width measurements. Plants that died are represented on graphs 633

with 0 cm height and 0 cm width. Number of dead is shown with n values. (D) Plant height 634

measurements for Lgn-R segregating 1:1 in Mo17 and in B73. A one-way ANOVA with a Tukey 635

Posthoc test (95%CI) showed that Lgn-R B73 is statistically more severe than Lgn-R Mo17 in 636

Albany, Ca (p < 1e-7). In Davis, the proportion of dead plants/genotype was found to be 637

statistically significant by z-test for Lgn-R B73 plants compared to Lgn-R Mo17 (z = -35.0, p < 638

0.00001). (E) Plant leaf width measurements for Lgn-R segregating 1:1 in Mo17 and in B73. A 639

one-way ANOVA with a Tukey Posthoc test (95%CI) showed that Lgn-R B73 is statistically more 640

severe than Lgn-R Mo17 in Albany, Ca (p < 1e-7). (F) Plant height measurements for Lgn-R 641

segregating 1:1 in NIL (Sol-B/Sol-M) and B73 (Sol-B/Sol-B) backgrounds. A one-way ANOVA 642

with a Tukey Posthoc test (95% CI) showed significant differences between Lgn-R NIL and Lgn-643

R B73 plants (p = 0.02). In Davis, the proportion of dead plants/genotype was found to be 644

statistically significant by z-test for Lgn-R NIL plants compared to Lgn-R B73 (z = -8.25, p < 645

0.00001) (G) Plant leaf width measurements for Lgn-R segregating 1:1 in NIL and B73 646

backgrounds. A one-way ANOVA with a Tukey Posthoc test (95% CI) showed significant 647

differences between Lgn-R NIL and Lgn-R B73 plants (p = 3.5e-6). (H) Percent of plants that 648

develop ears. A z-test found that the proportion of Lgn-R NIL plants with ears is significantly 649

greater than the proportion of Lgn-R B73 plants that form ears (z = -31.0, p < 0.00001). Error bars 650

represent standard error. (I, J) Plant height and leaf measurements for a family segregating for Lgn-651

R and Sol. A one way ANOVA with a Tukey Posthoc test (95% CI) found significant differences 652

between heterozygous and homozygous Lgn-R regardless of Sol genotype. (I) Within Lgn-R 653

heterozygotes, the NIL and Sol-M/-M plants have greater plant heights than their B73 siblings (p 654

< 0.007; p < 0.004) but there are not significant differences between the NIL and Sol-M/Sol-M 655

individuals. Among the Lgn-R homozygotes, the NIL has statistically shorter heights than Sol-656

M/Sol-M plants (p < 0.02) and is significantly taller than Sol-B/Sol-B (p < 0.007). (J) Within Lgn-657

23

R heterozygotes, the NIL and Sol-M/Sol-M plants have greater leaf widths than Sol-B/-B (p < 2e-658

4; p < 2e-6) but are not significantly different from each other. Within Lgn-R homozygotes, we 659

see a clear dosage effect with each copy of Sol-M, with heterozygotes significantly wider than Sol-660

B/Sol-B plants (p < 0.004) and signficantly narrower than Sol-M/Sol-M plants (p < 9e-4). (K) 661

Adaxial view of ligule region of Lgn-R heterozygotes in order from top to bottom of Sol-M/Sol-662

M, Sol-M/Sol-B, Sol-B/Sol-B. 663

664

Figure 2. Lgn-R plants survive the heat with the Sol-M modifier. 665

(A) Mature plant phenotypes of individuals segregating for Lgn-R and Sol-M in Davis, Ca. Dead666

plants (circled in red) are Lgn-R B73 and flank a Lgn-R NIL plant. (B) Height measurements of 667

individuals segregating for Lgn-R and Sol-M in cool (Albany, Ca) versus hot (Davis, Ca) climate. 668

Non-mutant siblings include both Sol genotypes. A two-tailed t-test showed that Lgn-R NIL plants 669

were significantly different between the two fields (t (49) = 9.1, p = 4e-12) as were Lgn-R B73 670

plants (t (47) = 15.2, p = 9e-20). (C) Top, from left to right: WT, Lgn-R NIL and Lgn-R B73 plants 671

at 30 days after planting in the cycling cool growth chamber (11-21°C). Below is same order in 672

the cycling hot growth chamber (15-32°C). (D) Mature plants 30 days post cycling hot growth 673

chamber treatment from left to right: non-mutant, Lgn-R NIL, Lgn-R B73. Only Lgn-R B73 plants 674

were unable to recover from heat treatment. (E) Adaxial view of the ligule of leaf six when plants 675

are grown at a constant 17°C. (F) Adaxial view of the ligule of leaf six when plants are grown at 676

a constant 30°C. (G) Plant heights and leaf widths for a family grown at a constant 17°C compared 677

to 30°C. A one way ANOVA revealed significant differences in height between the non-mutant 678

and Lgn-R B73 siblings at 30°C (p < 0.01) but not 17°C whereas significant differences in leaf 679

width were seen at both temperatures when comparing Lgn-R in B73 to the NIL (p < 1e-4; p < 5e-680

3) and non-mutant siblings. (H) The change in plant height and leaf width observed per genotype681

in 17°C conditions compared to 30°C conditions. Two-tailed t-tests showed that Lgn-R B73 plants 682

were most significantly altered between the two temperature regimes compared to both non-mutant 683

and NIL siblings in terms of both height (t(25) = 4.4 p < 2e-4 ; t(13) = 2.5 p < 0.03) and width 684

(t(25) = 2.3 p < 0.03; t (13) = 3.1 p < 8e-3). Lgn-R NIL lines were not significantly different from 685

their non-mutant siblings. (B,H) Error bars represent standard error. 686

687

24

Figure 3. Map position of Sol and analysis of the locus in NAM founder lines. 688

(A) Maize chromosome 1 with markers used for fine mapping of the Sol QTL. (B) Plant height of 689

maize inbreds crossed once to Lgn-R/+ in B73 and grown in Indiana. Error bars represent standard 690

error. (C-I) Plant height and leaf width comparisons for families segregating Lgn-R and Sol in our 691

cool (Albany, Ca) and hot (Davis, Ca) environments. Dead plants are represented on graphs with 692

0 cm height and 0 cm width and number per genotype, n, is shown. (C) Family segregating at Sol 693

locus for Mo17 and Ms71 alleles; heterozygotes are significantly rescued compared to 694

homozygotes. A one-way ANOVA with a Tukey Posthoc test (95% CI) found a significant 695

difference in leaf width in Albany, Ca (p = 1.3e-4). A z-test performed on the proportion of dead 696

in Davis, Ca also found significant differences (z = -11.8, p<0.00001). (D) Family segregating at 697

Sol locus for B73 and CML228 alleles; heterozygotes are not statistically different from 698

homozygotes at either location. (E) Family segregating at Sol for B73 and CML247; heterozygotes 699

are not statistically different from homozygotes at either location. (F) Family segregating at Sol 700

for B73 and Nc358; heterozygotes are not statistically different from homozygotes at either 701

location. (G) Family segregating at Sol for B73 and Nc350 alleles; heterozygotes display a 702

significant rescued phenotype at both locations compared to homozygotes. A one-way ANOVA 703

with a Tukey Posthoc test (95% CI) found a significant difference in leaf width in Albany, Ca (p 704

= 0.01). A z-test performed on the proportion of dead in Davis, Ca also found significant 705

differences (z = -27.5, p < 0.00001). (H) Family segregating at Sol for B73 and Tzi8 alleles; 706

heterozygotes display a significant rescued phenotype at both locations compared to homozygotes. 707

A one-way ANOVA with a Tukey Posthoc test (95% CI) found a significant difference in leaf 708

width (p = 1.5e-5) and plant height (p = 7.3e-4) in Albany, Ca. A z-test performed on the proportion 709

of dead in Davis, Ca also found significant differences (z = -11.8, p < 0.00001). (I) Family 710

segregating at Sol for B73 and M162W. A one-way ANOVA with a Tukey Posthoc Test (95% CI) 711

found a significant difference in leaf width (p = 0.01) in Albany, Ca. A z-test performed on the 712

proportion of dead heterozygotes compared to homozygotes in Davis, Ca also found significant 713

differences (z = -14.9, p < 0.00001). 714

715

Figure 4. Sequence analysis supports GRMZM2G075262 as Sol 716

25

(A) Partial alignment of SOL sequence in rescuing and non-rescuing maize inbred lines. The non-717

canonical ZF motif is in a purple box. Amino acid substitutions that segregate with ability to rescue 718

are in orange boxes. Indels that segregate with ability to rescue are in red boxes. The second ZF 719

domain is labeled on the cartoon and the site of EMS induced point mutation, Sol-G489E, is 720

marked on the cartoon with a blue box. (B) Normalized fold expression of syntenic genes in the 721

Sol QTL interval according to background. All treatments represent at least three biological 722

replicates. (C) RT-qPCR results examining Sol expression in mutant and non-mutant siblings in 723

severe (CML228, Ms71) and rescued (Nc350) backgrounds. Two-tailed t-tests found Sol 724

expression to be statistically higher only in the severe mutant backgrounds (CML228: t (2) = 29.9, 725

p = 0.001; Ms71: t (4) = 2.3, p = 0.05) (B,C) Error bars represent standard error. (D) Plant heights 726

and leaf widths at maturity in an EMS mutagenized Lgn-R population. The plant that was found to 727

contain a point mutation in GRMZM2G075262 is shown in red. (E) SOL protein tree. SOL and 728

EDR4 indicated with red stars. Tree generated with MEGA X using species: Zea mays, 729

Brachypodium distachyon, Sorghum bicolor, Oryza sativa japonica, Setaria italica, Arabidopsis 730

thaliana, Brassica rapa, Gossypium raimondii, Solanum lycopersicum, Populus trichocarpa. Note 731

that the bottom four branches represent collapsed nodes. 732

733

Figure 5. Sol exhibits expression and localization differences that are dependent on 734

background and treatment. 735

(A-C) SOL-B-YFP localization shown with Merged images (A), the YFP filter (B), and the DAPI 736

filter (C). (D-F) SOL-M-YFP localization shown with Merged images (D), the YFP filter (E), and 737

the DAPI filter (F). (G) Sol and Pr4 normalized fold expression as determined by RT-qPCR at four 738

different time points throughout chitin exposure. Two-tailed t-tests found Sol expression to be 739

statistically greater in the treatment versus the control at the 10 min (t (5) = 2.59, p = 0.05), 30 min 740

(t (3) = 2.3, p = 0.01), and 60 min (t (5) = 3.4, p = 0.02) timepoints. t-tests found Pr4 induction to 741

be significant at the 30 min (t (3) = 3.2, p = 0.05) and 60 min (t (3) = 3.5, p = 0.04) timepoints. (H) 742

Sol and Pr4 induction at 60-min time points in B73 and Lgn-R B73 backgrounds. Two tailed t-743

tests found significant Sol induction in non-mutant samples treated with flg22 (t (3) = 4.5, p = 744

0.02) and Lgn-R B73 samples treated with chitin showed significant repression of Sol (t (5) = 2.7, 745

p = 0.04). T-tests also found significant Pr4 induction in non-mutant B73 treated with flg22 (t (4) 746

26

= 4.3, p = 0.01) and Lgn-R B73 treated with chitin (t (4) = 8.3, p = 0.001) and flg22 (t (3) = 3.4, p 747

= 0.03.). (G,H) Error bars represent standard error and data represents a minimum of three 748

biological replicates. 749

750

Figure 6. Integrated quantitative RNA-seq and phosphoproteomics reveal a possible MAP 751

kinase signaling cascade that induces immune responses in Lgn-R. 752

(A-C) RNA-Seq of Lgn-R and nonmutant siblings revealed hundreds of significantly differentially 753

expressed genes. Here, we show changes in transcript abundance as fold-change in FPKM 754

(fragments per kilobase of transcript per million mapped reads) in Lgn-R compared to wild-type 755

(WT). Among these genes, the most significantly overrepresented categories are genes encoding 756

WRKY transcription factors (A), PATHOGENESIS-RELATED (PR) proteins that are typically 757

induced when plants detect pathogens (B), and receptors, including several families of receptor-758

like kinases and Nod-like receptors that are involved in sensing and responding to pathogen 759

infection (C). (D) LGN resides at the plasma membrane and signals through its kinase activity 760

to promote leaf development and block a MAP-kinase immunity cascade. In the presence of the 761

Lgn-R mutation, leaf development is compromised and the MAPK cascade is activated leading to 762

a pathogen-triggered immunity (PTI) response which can be dampened by cool temperatures or 763

the presence of Sol-M. 764

765

REFERENCES 766

1. Rodríguez GR, Kim HJ, & Van Der Knaap E (2013) Mapping of two suppressors of 767

OVATE (sov) loci in tomato. Heredity 111(3):256. 768

2. Gibert JM, Marcellini S, David J, Schlötterer C, & Simpson P (2005) A major bristle QTL 769

from a selected population of Drosophila uncovers the zinc-finger transcription factor 770

poils-au-dos, a repressor of achaete-scute. Dev Biol (288):194-205. 771

3. Buckler E, Gaut B, & MD M (2006) Molecular and functional diversity of maize. Curr 772

Opin Plant Biol 9:172-176. 773

4. Brunner S, Fengler K, Morgante M, Tingey S, & Rafalski A (2005) Evolution of DNA 774

sequence nonhomologies among maize inbreds. The Plant Cell 17:343-360. 775

27

5. Babu P, Agrawal P, Saha S, & Gupta H (2015) Mapping QTLs for opaque2 modifiers 776

influencing the tryptophan content in quality protein maize using genomic and candidate 777

gene-based SSRs of lysine and tryptophan metabolic pathway. . Plant Cell Rep 34:37-45. 778

6. Penning B, Johal G, & McMullen M (2004) A major suppressor of cell death, slm1, 779

modifies the expression of the maize (Zea mays L.) lesion mimic mutation les23. Genome 780

47:961-969. 781

7. Xing A, et al. (2014) A pair of homoeolog ClpP5 genes underlies a virescent yellow‐like 782

mutant and its modifier in maize. The Plant Journal 79(2):192-205. 783

8. Moon J, Candela H, & Hake S (2013) The Liguleless narrow mutation affects proximal-784

distal signaling and leaf growth. Development 140:405-412. 785

9. Chaffey N (2000) Physiological anatomy and function of the membranous grass ligule. 786

New Phytol. 146:5-21. 787

10. Buescher EM, Moon J, Runkel A, Hake S, & Dilkes BP (2014) Natural variation at 788

sympathy for the ligule controls penetrance of the semidominant Liguleless narrow-R 789

mutation in Zea mays. Genes, Genomes, Genetics 4(12):2297-2306. 790

11. Lee M, et al. (2002) Expanding the genetic map of maize with the intermated B73 X Mo17 791

(IBM) population. Plant Mol Biol 48:453-461. 792

12. van Wersch R, Li X, & Zhang Y (2016) Mighty dwarfs: Arabidopsis autoimmune mutants 793

and their usages in genetic dissection of plant immunity. Frontiers in plant science 7:1717. 794

13. Wu G, et al. (2015) ENHANCED DISEASE RESISTANCE4 associates with CLATHRIN 795

HEAVY CHAIN2 and modulates plant immunity by regulating relocation of EDR1 in 796

Arabidopsis. The Plant Cell 27(3):857-873. 797

14. Walley JW, et al. (2016) Integration of omic networks in a developmental atlas of maize. 798

Science 353(6301):814-818. 799

15. Lyons E & Freeling M (2008) How to usefully compare homologous plant genes and 800

chromosomes as DNA sequences. PLant J 53(4):661-673. 801

16. McMullen MD, et al. (2009) Genetic Properties Of The Maize Nested Association 802

Mapping Population. Science 325:737-740. 803

17. Zhao W, et al. (2006) Panzea: a database and resource for molecular and functional 804

diversity in the maize genome. Nucleic Acids Research 34(suppl_1):D752-D757. 805

28

18. Winter D, et al. (2007) An “Electronic Fluorescent Pictograph” browser for exploring and 806

analyzing large-scale biological data sets. PloS one 2(8):e718. 807

19. Zhang X, Valdés-López O, Arellano C, Stacey G, & Balint-Kurti P (2017) Genetic 808

dissection of the maize (Zea mays L.) MAMP response. Theoretical and Applied Genetics 809

130(6):1155-1168. 810

20. Cheng C, et al. (2013) Plant immune response to pathogens differs with changing 811

temperatures. Nature communications 4:2530-2530. 812

21. Park HC, et al. (2011) Arabidopsis MAP kinase phosphatase 1 is phosphorylated and 813

activated by its substrate AtMPK6. Plant cell reports 30(8):1523-1531. 814

22. Huot B, Yao J, Montgomery BL, & He SY (2014) Growth–defense tradeoffs in plants: a 815

balancing act to optimize fitness. Molecular plant 7(8):1267-1287. 816

23. Hu G, Richter TE, Hulbert SH, & Pryor T (1996) Disease lesion mimicry caused by 817

mutations in the rust resistance gene rp1. The Plant Cell 8(8):1367-1376. 818

24. Hua J, Grisafi P, Cheng S-H, & Fink GR (2001) Plant growth homeostasis is controlled by 819

the Arabidopsis BON1 and BAP1 genes. Genes & Development 15(17):2263-2272. 820

25. Jambunathan N, Siani JM, & McNellis TW (2001) A humidity-sensitive Arabidopsis 821

copine mutant exhibits precocious cell death and increased disease resistance. The Plant 822

Cell 13(10):2225-2240. 823

26. Zhang Y, Goritschnig S, Dong X, & Li X (2003) A gain-of-function mutation in a plant 824

disease resistance gene leads to constitutive activation of downstream signal transduction 825

pathways in suppressor of npr1-1, constitutive 1. The Plant Cell 15(11):2636-2646. 826

27. Yang S & Hua J (2004) A haplotype-specific Resistance gene regulated by BONZAI1 827

mediates temperature-dependent growth control in Arabidopsis. The Plant Cell 828

16(4):1060-1071. 829

28. Negeri A, et al. (2013) Characterization of temperature and light effects on the defense 830

response phenotypes associated with the maize Rp1-D21 autoactive resistance gene. BMC 831

plant biology 13(1):106. 832

29. Huang X, Li J, Bao F, Zhang X, & Yang S (2010) A gain-of-function mutation in the 833

Arabidopsis disease resistance gene RPP4 confers sensitivity to low temperature. Plant 834

physiology:pp. 110.157610. 835

29

30. Yang H, et al. (2010) A mutant CHS3 protein with TIR‐NB‐LRR‐LIM domains modulates836

growth, cell death and freezing tolerance in a temperature‐dependent manner in837

Arabidopsis. The Plant Journal 63(2):283-296.838

31. Hua J (2013) Modulation of plant immunity by light, circadian rhythm, and temperature.839

Current opinion in plant biology 16(4):406-413. 840

32. Huot B, et al. (2017) Dual impact of elevated temperature on plant defence and bacterial841

virulence in Arabidopsis. Nature communications 8(1):1808. 842

33. Ning J, Zhang B, Wang N, Zhou Y, & Xiong L (2011) Increased leaf angle1, a Raf-like843

MAPKKK that interacts with a nuclear protein family, regulates mechanical tissue 844

formation in the lamina joint of rice. The Plant Cell 23(12):4334-4347. 845

34. Hufford MB, et al. (2012) Comparative population genomics of maize domestication and846

improvement. Nature Genetics 44(7):808-811. 847

35. Edgar RC (2004) MUSCLE: multiple sequence alignment with high accuracy and high848

throughput. Nucleic acids research 32(5):1792-1797. 849

36. Kumar S, Stecher G, Li M, Knyaz C, & Tamura K (2018) MEGA X: Molecular850

Evolutionary Genetics Analysis across Computing Platforms. Molecular biology and 851

evolution 35(6):1547-1549. 852

37. Schäffer AA, et al. (2001) Improving the accuracy of PSI-BLAST protein database853

searches with composition-based statistics and other refinements. Nucleic acids research 854

29(14):2994-3005. 855

38. Ramakers C, Ruijter JM, Deprez RHL, & Moorman AF (2003) Assumption-free analysis856

of quantitative real-time polymerase chain reaction (PCR) data. Neuroscience letters 857

339(1):62-66. 858

39. Ewels P, Magnusson M, Lundin S, & Käller M (2016) MultiQC: summarize analysis859

results for multiple tools and samples in a single report. Bioinformatics 32(19):3047-3048. 860

40. Kim D, Langmead B, & Salzberg SL (2015) HISAT: a fast spliced aligner with low861

memory requirements. Nature methods 12(4):357. 862

41. Anders S, Pyl PT, & Huber W (2015) HTSeq—a Python framework to work with high-863

throughput sequencing data. Bioinformatics 31(2):166-169. 864

42. Merchant N, et al. (2016) The iPlant collaborative: cyberinfrastructure for enabling data to865

discovery for the life sciences. PLoS biology 14(1):e1002342. 866

30

43. McCarthy DJ, Chen Y, & Smyth GK (2012) Differential expression analysis of multifactor 867

RNA-Seq experiments with respect to biological variation. Nucleic acids research 868

40(10):4288-4297. 869

44. Tian T, et al. (2017) agriGO v2. 0: a GO analysis toolkit for the agricultural community, 870

2017 update. Nucleic acids research 45(W1):W122-W129. 871

45. Earley KW, et al. (2006) Gateway‐compatible vectors for plant functional genomics and 872

proteomics. The Plant Journal 45(4):616-629. 873

46. Bolduc N & Hake S (2009) The Maize Transcription Factor KNOTTED1 Directly 874

Regulates the Gibberellin Catabolism Gene ga2ox1. Plant Cell 21:1647-1658. 875

876

WT Lgn-R Mo17

Lgn-R NIL

Lgn-R B73

A B C D E

Wt Mo17 WT B73Mo17

Lgn-R/+NIL

Lgn-R/+B73

Lgn-R/+

Figure 1

��

�

���

��

�

�

�

�

�

�

��

� ��

� �

��

��

���

��

���

���

�����

�

���������

��

����

���

��

��

�

��

�

����

��

��

����

�

��

�

���

����

��

��

�

��

�

�

��

��

�

�

�

��

�

���

�

�

�

� �� ���

�

������

�

��

� �

��

�

��

�

�

��

�

Albany, Ca Davis, Ca

Albany, Ca Davis, Ca

Hei

ght (

cm)

Leaf

wid

th (c

m)

Non-MutantB73

Lgn-R B73

Lgn-R Mo17

Non-MutantMo17

Non-Mutant

Lgn-RNIL

Lgn-RB73

*D

E

n=2 n=3

n=2 n=3

�

� �

�

�

�

�

�

�

�

��

��

���

��

�

� ���������

�

�

��

��

����

�

���

�

��

���

��� �����

�

�

�

�

�

�

��

�

�

�

�

�

�

�� �� ��

���

�

�

��

�

�

�

�

�

�

��

�

�

�

����� ������

�

�

�

�

��

���

��

���

� ������

���

�

�

�

�

����

���

�

�

�

��

�

��

�

�

�

�

��

�

����

�

�

�

�

��

��

�

��

�

�

�

���

�

�

���� ���� ������

��

��

�

�

�

���

���

��

�

0

4

8

12

n=13 n=14

�

�

�

����

�

��

�

�� �

�

��

���

��

�� �

�

�

�

�

��

�

��

����

�

�

�

��

��� ���

�

����� ��

��

��

�� �����

�����

�

���

�

�

�

�

�

�

�

�

�

�

��

�

�

��

�

�

�

����� ����� �

��

�

�

�

�

���

�

�� ��

�

����

���

��

���

���

���

���

���

�

�

���

�

�

���

�

� �

�

�

�

�

���

�

��

��

����

����

�

����� ������� ��

����

��

�

��� �

���

��

0

100

200

n=13 n=14

250

***

*********

***

******

***

***

Lgn-R NIL

Lgn-R B73

***

100

% w

ith e

ars

B

C

Albany, Ca Davis, Ca

Albany, Ca Davis, Ca

Hei

ght (

cm)

F

0

100

200

250

Leaf

wid

th (c

m)

G

0

4

8

12

HLgn-R

Sol-B/-B

Lgn-R Sol-M/-M

Non-Mutant

Lgn-RSol-M/-B

I

���

��

����

�������������

����

������

��

��

�

���

�

��

��

�

����

��

�

����

�

��

��

���

�

�

��

��

�

��

�

�����

��

���

�

��������

�����

����

0

100

200

Lgn-R/Lgn-R

Lgn-R/+

Non-Mutant

**

*

�

�

�����

������

����

�

��

������������

�����

����

���

���

��

�

����

��

��������

�

����

�

�

��

�����

�

�

�����

��

����

��

������

0

2

4

6

8

Leaf

wid

th (c

m)

Lgn-R/Lgn-R

Lgn-R/+

Non-Mutant

******

**

J

50

0

Hei

ght (

cm)

***

******

K

Sol-M/Sol-M

Sol-M/Sol-B

Sol-B/Sol-B

Figure 1. The Sol-M modifier rescues the Lgn-R phenotype. (A) Mature plant phenotypes pictured from left to right: Mo17, B73, Lgn-R Mo17, Lgn-R NIL, Lgn-R B73. Ears are circled inred. (B) Adaxial view of ligules from leaf six. Lgn-R B73 ligule in red box. (C) Abaxial view of ligules from leaf six in sameorder as (B). Auricles marked by red angles. (D-G) Plant height and leaf width measurements. Plants that died are repre-sented on graphs with 0 cm height and 0 cm width. Number of dead is shown with n values. (D) Plant height measurementsfor Lgn-R segregating 1:1 in Mo17 and in B73. A one-way ANOVA with a Tukey Posthoc test (95%CI) showed that Lgn-RB73 is statistically more severe than Lgn-R Mo17 in Albany, Ca (p < 1e-7). In Davis, the proportion of dead plants/genotypewas found to be statistically significant by z-test for Lgn-R B73 plants compared to Lgn-R Mo17 (z = -35.0, p < 0.00001). (E)Plant leaf width measurements for Lgn-R segregating 1:1 in Mo17 and in B73. A one-way ANOVA with a Tukey Posthoc test(95%CI) showed that Lgn-R B73 is statistically more severe than Lgn-R Mo17 in Albany, Ca (p < 1e-7). (F) Plant heightmeasurements for Lgn-R segregating 1:1 in NIL (Sol-B/Sol-M) and B73 (Sol-B/Sol-B) backgrounds. A one-way ANOVA witha Tukey Posthoc test (95% CI) showed significant differences between Lgn-R NIL and Lgn-R B73 plants (p = 0.02). In Davis,the proportion of dead plants/genotype was found to be statistically significant by z-test for Lgn-R NIL plants compared toLgn-R B73 (z = -8.25, p < 0.00001) (G) Plant leaf width measurements for Lgn-R segregating 1:1 in NIL and B73 back-grounds. A one-way ANOVA with a Tukey Posthoc test (95% CI) showed significant differences between Lgn-R NIL andLgn-R B73 plants (p = 3.5e-6). (H) Percent of plants that develop ears. A z-test found that the proportion of Lgn-R NIL plantswith ears is significantly greater than the proportion of Lgn-R B73 plants that form ears (z = -31.0, p < 0.00001). Error barsrepresent standard error. (I, J) Plant height and leaf measurements for a family segregating for Lgn-R and Sol. A one wayANOVA with a Tukey Posthoc test (95% CI) found significant differences between heterozygous and homozygous Lgn-Rregardless of Sol genotype. (I) Within Lgn-R heterozygotes, the NIL and Sol-M/-M plants have greater plant heights thantheir B73 siblings (p < 0.007; p < 0.004) but there are not significant differences between the NIL and Sol-M/Sol-M individu-als. Among the Lgn-R homozygotes, the NIL has statistically shorter heights than Sol-M/Sol-M plants (p < 0.02) and issignificantly taller than Sol-B/Sol-B (p < 0.007). (J) Within Lgn-R heterozygotes, the NIL and Sol-M/Sol-M plants have great-er leaf widths than Sol-B/-B (p < 2e-4; p < 2e-6) but are not significantly different from each other. Within Lgn-R homozy-gotes, we see a clear dosage effect with each copy of Sol-M, with heterozygotes significantly wider than Sol-B/Sol-B plants(p < 0.004) and signficantly narrower than Sol-M/Sol-M plants (p < 9e-4). (K) Adaxial view of ligule region of Lgn-Rheterozygotes in order from top to bottom of Sol-M/Sol-M, Sol-M/Sol-B, Sol-B/Sol-B.

Figure 2. Lgn-R plants survive the heat with the Sol-M modifier. (A) Mature plant phenotypes of individuals segregating for Lgn-R and Sol-M in Davis, Ca. Dead plants (circled in red) areLgn-R B73 and flank a Lgn-R NIL plant. (B) Height measurements of individuals segregating for Lgn-R and Sol-M in cool(Albany, Ca) versus hot (Davis, Ca) climate. Non-mutant siblings include both Sol genotypes. A two-tailed t-test showedthat Lgn-R NIL plants were significantly different between the two fields (t (49) = 9.1, p = 4e-12) as were Lgn-R B73 plants(t (47) = 15.2, p = 9e-20). (C) Top, from left to right: WT, Lgn-R NIL and Lgn-R B73 plants at 30 days after planting in thecycling cool growth chamber (11-21°C). Below is same order in the cycling hot growth chamber (15-32°C). (D) Matureplants 30 days post cycling hot growth chamber treatment from left to right: non-mutant, Lgn-R NIL, Lgn-R B73. OnlyLgn-R B73 plants were unable to recover from heat treatment. (E) Adaxial view of the ligule of leaf six when plants aregrown at a constant 17°C. (F) Adaxial view of the ligule of leaf six when plants are grown at a constant 30°C. (G) Plantheights and leaf widths for a family grown at a constant 17°C compared to 30°C. A one way ANOVA revealed significantdifferences in height between the non-mutant and Lgn-R B73 siblings at 30°C (p < 0.01) but not 17°C whereas significantdifferences in leaf width were seen at both temperatures when comparing Lgn-R in B73 to the NIL (p < 1e-4; p < 5e-3)and non-mutant siblings. (H) The change in plant height and leaf width observed per genotype in 17°C conditions com-pared to 30°C conditions. Two-tailed t-tests showed that Lgn-R B73 plants were most significantly altered between thetwo temperature regimes compared to both non-mutant and NIL siblings in terms of both height (t(25) = 4.4 p < 2e-4 ; t(13)= 2.5 p < 0.03) and width (t(25) = 2.3 p < 0.03; t (13) = 3.1 p < 8e-3). Lgn-R NIL lines were not significantly different fromtheir non-mutant siblings. (B,H) Error bars represent standard error.

012345678

Heig

ht (c

m)

Wid

th (c

m)

C

AFigure 2

B

Non-mutant

Lgn-RNIL

Lgn-RB73

Plan

t Heig

ht (c

m)

AlbanyField

Davis Field

*

*

C

Non-mutantsiblings

D E 17°C

F 30°C

Non-mutantLgn-RB73

Non-mutant

Lgn-R

B73

∆Heig

ht (c

m)

Non-Mutant

Lgn-R B73

Lgn-R NIL

00.10.20.30.40.50.6 */*

**/*����

��

����

�

�

�

�

�

��

�

���

�

�

�

����

���

�

��

�

���

� �

�

���

�

�

�

�

��

�

��

�

�

�

��

�

��

��

�

�

�

�

�

�

�

�

��

�

��

�

���

�

� �10

15

250

200

150

100

50

0

17C 30C

���

��

��

�

����

�

�

�

�

��

�� �

�����

�

��

�

���

�

�

��

�

�

��� �

��

�

�

����

�

���

�

�

�

�

����

����

����

�

�

�

�

�

�

��

�

�

��

1.00

1.25

1.50

1.75

Wid

th (c

m)

∆

*

G H

Non-Mutant

Lgn-R B73

Lgn-R NIL

*

***

***

Figure 3A

bin 1.04

CHR 1

53.1 54.1 55.1 56.6 58.5 60.8 61.7 70.8Map Position

(Mb)GRMZM2G075262GRMZM2G072892GRMZM2G049211GRMZM2G119850

B

�

��

���

��

����

���

���

�

������

�� ��

�

��

�

��������� �

���

��

��

���

��

���

�

����

����

����

����� ��������

����

��

���� �

�

�

�

��� ��

�

�

�

��

�

� �

��

���

��

�

�����

�

�

���

��������

��

�

� ���������

�

��

���

� ��

���

�

��

����

�

���

�

�

�

�

��

��

� ��

������

���

�

�

��� �

����

�

�

�

��

��

��

�

�

�

���

�

�������������

����

�

��

�

�

� �� ��

�

�������

�

�

�

��

�

��

��

������

� �����

�

�

��

����

���

�����������

�

���

Nc350

Albany, Ca Davis, Ca

Hei

ght (

cm)

Leaf

wid

th (c

m)

Non-Mutant

Lgn-R Sol-Nc350/Sol-BLgn-R Sol-B/Sol-B

�

���

��

� ��

� ���

��

���

��

���

��

��

�

���������

�

����

�������

���

�

��

�

� �

����

���� �

� ����

�����

���������� �����

��

�����

Tzi8

�����

��

���

�������

���

�� ��

�

���

� ���

�

��

��� �����

����

��

�

��

��

�

�

��

��

��

��

�

���

���

��

����

�

��� ��������

���

��

� ����

Albany, Ca Davis, Ca

Lgn-R Sol-M162W/Sol-B

�

� ���

�

�

��

��

���

��������

�� ���

�

�

����

�

��

�

�

�

��

�

�

����

050

100150200250

����

�

�

�

�� ��

�

��

����

�

�

�

���

���

�

�

�

���

�

���

�

�

��

�

�����

Lgn-R Sol-CML247/Sol-B Lgn-R Sol-B/Sol-B

Albany, Ca Davis, Ca

Ms71

G H

C

�

�

�� �

�

�

��

�

�

��� �

��

�

�

����

��

�

�

�

�� ���

� ���� ������ ����� ��

���� �

0

3

6

9

12�

��

�

�

�

�

��

�

��

�

����

��

�

�

�

��

�

�

�

�

����

�

������ ���� ��������

��

�

��

Albany, Ca Davis, Ca

CML228

Lgn-R Sol-Nc358/Sol-B

n=1n=1 n=3 n=11n=4

n=2 n=6n=10n=3

n=1

D

n=1n=1 n=3n=11

n=4

n=3n=1 n=2 n=6n=10

050

100150200250

050

100150200250

050

100150200250

0

3

6

9

12

0

3

6

9

12

0

3

6

9

12

Lgn-R Sol-B/Sol-B

Lgn-R Sol-B/Sol-B

TIDP4672 (11)UMC2145 (5)bnlg2238 (2)IDP1489 (4)

IDP8320 (6)

IDP8458 (8)IDP182 (11)

bnlg1811 (18)

***

****

***

***

***

***

***

***

***

050

100150200250

050

100150200250

0

3

6

9

12

0

3

6

9

120

50100150200250

��

�

�

�

�

��

�

��

�

����

��

�

�

�

��

�

�

�

�

����

������� �����������

��

�

��

�

���

�

�

��

�

�

����

��

�

�

����

� �

�

�

�

�� ��

����� �������������

�����

0

3

6

9

12

����

��

�������

���

����

���

�

��

��

�

�������

�

�

�

�

��

��

���

�

��

�

�� ��

��

��� �

��

��

��

�

�

�

��

�����

�

�

�

�

��

�

��

�

��� �

�

�

�

�

�

�

�

�

�

� �

�

��

��

��

�

�

�

�

�

�

�

� �

�

�

�

��

�

���

�

�

����� ��

����

����

�

�

��

��

�

���

��

�

�

�

�

�����

�

�

��

�

����

�� ��

���

�

�

���

�

���

��

CML247

Hei

ght (

cm)

Leaf

wid

th (c

m)

Non-Mutant

Albany, Ca Davis, Ca

Lgn-R Sol-Tzi8/Sol-BLgn-R Sol-B/Sol-B

Lgn-R Sol-Ms71/Sol-M Lgn-R Sol-Ms71/Sol-Ms71

Albany, Ca Davis, Ca

Nc358

Albany, Ca Davis, Ca

M162W

Lgn-R Sol-CML228/Sol-B Lgn-R Sol-B/Sol-B

F

I

E

n=1 n=11n=3 n=3n=1

n=6 n=10

n=1 n=11n=3 n=3n=1

n=6n=10

***

*

***

0

50

100

150

200

250

300

350

Plan

t Hei

ght (

cm)

Non-mutant Lgn-R/+

Figure 3. Map position of Sol and analysis of the locus in NAM founder lines. (A) Maize chromosome 1 with markers used for fine mapping of the Sol QTL. (B) Plant height of maize inbreds crossedonce to Lgn-R/+ in B73 and grown in Indiana. Error bars represent standard error. (C-I) Plant height and leaf width com-parisons for families segregating Lgn-R and Sol in our cool (Albany, Ca) and hot (Davis, Ca) environments. Dead plantsare represented on graphs with 0 cm height and 0 cm width and number per genotype, n, is shown. (C) Family segregatingat Sol locus for Mo17 and Ms71 alleles; heterozygotes are significantly rescued compared to homozygotes. A one-wayANOVA with a Tukey Posthoc test (95% CI) found a significant difference in leaf width in Albany, Ca (p = 1.3e-4). A z-testperformed on the proportion of dead in Davis, Ca also found significant differences (z = -11.8, p<0.00001). (D) Familysegregating at Sol locus for B73 and CML228 alleles; heterozygotes are not statistically different from homozygotes ateither location. (E) Family segregating at Sol for B73 and CML247; heterozygotes are not statistically different from homo-zygotes at either location. (F) Family segregating at Sol for B73 and Nc358; heterozygotes are not statistically differentfrom homozygotes at either location. (G) Family segregating at Sol for B73 and Nc350 alleles; heterozygotes display asignificant rescued phenotype at both locations compared to homozygotes. A one-way ANOVA with a Tukey Posthoc test(95% CI) found a significant difference in leaf width in Albany, Ca (p = 0.01). A z-test performed on the proportion of deadin Davis, Ca also found significant differences (z = -27.5, p < 0.00001). (H) Family segregating at Sol for B73 and Tzi8alleles; heterozygotes display a significant rescued phenotype at both locations compared to homozygotes. A one-wayANOVA with a Tukey Posthoc test (95% CI) found a significant difference in leaf width (p = 1.5e-5) and plant height (p =7.3e-4) in Albany, Ca. A z-test performed on the proportion of dead in Davis, Ca also found significant differences (z =-11.8, p < 0.00001). (I) Family segregating at Sol for B73 and M162W. A one-way ANOVA with a Tukey Posthoc Test (95%CI) found a significant difference in leaf width (p = 0.01) in Albany, Ca. A z-test performed on the proportion of dead hetero-zygotes compared to homozygotes in Davis, Ca also found significant differences (z = -14.9, p < 0.00001).

Figure 4A

Non-canonical ZF and Segregating

AA Variation

ZF Domain DUF3133

M162W MASTEGFRLVRCPKCLNILPEPPNVTVYKCGGCGTTLR-AKTRASGGQQVA--AKQG-RQ Nc358 MASTEGFRLVRCPKCLNILPEPPNVTVYKCGGCGTTLR-AKTRASEGQQVA--AKQGRRQ Ms71 MASTEGFRLVRCPKCLNILPEPPNVTVYKCGGCGTTLR-AKTRASGGQQVAATAKQG-RQ B73 MASTEGFRLVRCPKCLNILPEPPNVTVYKCGGCGTTLR-AKTRASGGQQVAATAKQG-RQ Mo17 MASTEGFRLVRCPKCLNILPEPPNVTVYKCGGCGTTLRCAKTRASGGQQVA--AKQG-RQ Tzi8 MASTEGFRLVRCPKCLNILPEPXNVTVYKCGGCGTTLR-AKTRASGGQQVA--AKQG-RQ CML247 MASTEGFRLVRCPKCLNILPEPPNVTVYKCGGCGTTLS-AKTRASGGQQVA--AKQG-RQ CML228 MASTEGFRLVRCPKCLNILPEPPNVTVYKCGGCGTTLR-AKTRASGGQQVA--AKQG-RQ Nc350 MASTEGFRLVRCPKCLNILPEPPNVTVYKCGGCGTTLRCAKTRASGGQQVA--AKQG-RQ ********************** ************** ****** ***** **** **

M162W DSDCYSVATAVSNGVPRQNRDQ---GAVTESSFDADVA----------SGENGGGMLAGQ Nc358 DSDSYSVATAVSSGVPRQNRNQGSIGAVTESSFDADVPSAEHGSNGARSGENGGGMLAGQ Ms71 DSDSYSVATAVSSGVPRQNRDQGSIGAVTESSFDADVASAEHGSNGARSGENGGGMLAGQB73 DSDSYSVATAVSSGVPRQNRDQGSIGAVTESSFDADVASAEHGSNGARSGENGGGMLAGQMo17 DSDCYSVATAVSNGVPRQNRDQ---GAVTESSFDADVA----------SGENGGGMLAGQ Tzi8 DSDCYSVATAVSNGVPRQNRDQ---GAVTESSFDADVA----------SGENGGGMLAGQ CML247 DSDCYSVATAVSNGVPRQNRDQ---GAVTESSFDADVA----------SGENGGGMLAGQ CML228 DSDCYSVATAVSNGVPRQNRDQ---GAVTESSFDADVA----------SGENGGGMLAGQ Nc350 DSDCYSVATAVSNGVPRQNRDQ---GAVTESSFDADVA----------SGENGGGMLAGQ ***.********.*******:* ************. ************

M162W NARFEGQD-NTSSRMEGPAGQTRLNASA---DSGGTENHAVQQTAEKCRVSGHDDDTECH Nc358 NVHFEGQDNNTSSRMEGPAGQTPLNANATYLNSGGTKNHTVQQTAKKCRVSGHVDDTECH Ms71 NVHFEGQDNNTSSRMEGPAGQTRLNANATYLDSGGTENHTVQQTAEKCRVSGHVDDTECHB73 NVHFEGQDNNTSSRMEGPAGQTRLNANATYLDSGGTENHTVQQTAEKCRVSGHVDDTECHMo17 NARFEGQD-NTSSRMEGPAGQTRLNASA---NSGGTENHAVQQTAEKCRVSGHDDDTECH Tzi8 NAHFEGQD-NTSSRMEGPAGQTRLNASA---DSGGTENHAVQQTAEKCRVSGHDDDTECH CML247 NARFEGQD-NTSSRMEGPAGQTRLNASA---DSGGTENHAVQQTAEKCRVSGHDDDTECH CML228 NARFEGQD-NTSSRMEGPAGQTRLNASA---DSGGTENHAVQQTAEKCRVSGHDDDTECH Nc350 NARFEGQD-NTSSRMEGPAGQTRLNASA---DSGGTENHAVQQTAEKCRVSGHDDDTECH *..***** ************* ***.* :****:**:*****:******* ******

LNTSEDEMFPSETSKAAVGMQDPEQKKEAGGTEHAANKKSHLVRALSRSCDLGPSINSTD

PGQKKEAGGTEHAANKKSHLVRALSRSCDLGSSMNLTD

RSCDLGPSINSTD

:.********************* *****************************.*:* **

M162W FHSARGSLQSKSFRASAPLQSKIMSTVDELKGDLSELFSKPADCKPR--PHPHPPRPSKR Nc358 FHSARTSLQSKSFRASAPLQSKIMSTVDELKGDLSELFSKPADCKPR--PHPHPPRPSKR Ms71 FHSARASLQSKSFRASAPLQSKIMSTVDELKGDLSELFSKPADCKPRPHPHPHPPRPSKRB73 FHSARASLQSKSFRASAPLQSKIMSTVDELKGDLSEMFSKPADCKPR--PHPHPPRPSKR Mo17 FHSARASLQSKSFRASAPLQSKIMSTVDELKGDLSEMFSKPADCKPR--PHPHPPRPSKR Tzi8 FHSARTSLQSKSFRASAPLQSKIMSTVDELKGDLSELFSKPADCKPR--PHPHPPRPSKR CML247 FHSARTSLQSKSFRASAPLQSKIMSTVDELKGDLSELFSKPADCKPR--PHPHPPRPSKR CML228 FHSARTSLQSKSFRASAPLQSKIMSTVDELKGDLSELFSKPADCKPR--PHPHPPRPSKR Nc350 FHSARTSLQSKSFRASAPLQSKIMSTVDELKGDLSELFSKPADCKPR--PHPHPPRPSKR ***** ******************************:********** ***********

M162W DGGRTARAAVTSSAPLAAYRPPAEHSGYVSRSRLSRSAQVLPPPRRGLPSRGIAGTGVYPNc358 DGGRTARAAVTSSAPLAAYRPPAEHSGYVSRSRLSRSAQVLPPPRRGLPSLRYRRHRVYP

M162W MASTEGFRLVRCPKCLNILPEPPNVTVYKCGGCGTTLR-AKTRASGGQQVA--AKQG-RQ Nc358 MASTEGFRLVRCPKCLNILPEPPNVTVYKCGGCGTTLR-AKTRASEGQQVA--AKQGRRQ Ms71 MASTEGFRLVRCPKCLNILPEPPNVTVYKCGGCGTTLR-AKTRASGGQQVAATAKQG-RQ B73 MASTEGFRLVRCPKCLNILPEPPNVTVYKCGGCGTTLR-AKTRASGGQQVAATAKQG-RQ Mo17 MASTEGFRLVRCPKCLNILPEPPNVTVYKCGGCGTTLRCAKTRASGGQQVA--AKQG-RQ Tzi8 MASTEGFRLVRCPKCLNILPEPXNVTVYKCGGCGTTLR-AKTRASGGQQVA--AKQG-RQ CML247 MASTEGFRLVRCPKCLNILPEPPNVTVYKCGGCGTTLS-AKTRASGGQQVA--AKQG-RQ CML228 MASTEGFRLVRCPKCLNILPEPPNVTVYKCGGCGTTLR-AKTRASGGQQVA--AKQG-RQ Nc350 MASTEGFRLVRCPKCLNILPEPPNVTVYKCGGCGTTLRCAKTRASGGQQVA--AKQG-RQ ********************** ************** ****** ***** **** **

M162W DSDCYSVATAVSNGVPRQNRDQ---GAVTESSFDADVA----------SGENGGGMLAGQ Nc358 DSDSYSVATAVSSGVPRQNRNQGSIGAVTESSFDADVPSAEHGSNGARSGENGGGMLAGQ Ms71 DSDSYSVATAVSSGVPRQNRDQGSIGAVTESSFDADVASAEHGSNGARSGENGGGMLAGQ B73 DSDSYSVATAVSSGVPRQNRDQGSIGAVTESSFDADVASAEHGSNGARSGENGGGMLAGQ Mo17 DSDCYSVATAVSNGVPRQNRDQ---GAVTESSFDADVA----------SGENGGGMLAGQ Tzi8 DSDCYSVATAVSNGVPRQNRDQ---GAVTESSFDADVA----------SGENGGGMLAGQ CML247 DSDCYSVATAVSNGVPRQNRDQ---GAVTESSFDADVA----------SGENGGGMLAGQ CML228 DSDCYSVATAVSNGVPRQNRDQ---GAVTESSFDADVA----------SGENGGGMLAGQ Nc350 DSDCYSVATAVSNGVPRQNRDQ---GAVTESSFDADVA----------SGENGGGMLAGQ ***.********.*******:* ************. ************

M162W NARFEGQD-NTSSRMEGPAGQTRLNASA---DSGGTENHAVQQTAEKCRVSGHDDDTECH Nc358 NVHFEGQDNNTSSRMEGPAGQTPLNANATYLNSGGTKNHTVQQTAKKCRVSGHVDDTECH Ms71 NVHFEGQDNNTSSRMEGPAGQTRLNANATYLDSGGTENHTVQQTAEKCRVSGHVDDTECH B73 NVHFEGQDNNTSSRMEGPAGQTRLNANATYLDSGGTENHTVQQTAEKCRVSGHVDDTECH Mo17 NARFEGQD-NTSSRMEGPAGQTRLNASA---NSGGTENHAVQQTAEKCRVSGHDDDTECH Tzi8 NAHFEGQD-NTSSRMEGPAGQTRLNASA---DSGGTENHAVQQTAEKCRVSGHDDDTECH CML247 NARFEGQD-NTSSRMEGPAGQTRLNASA---DSGGTENHAVQQTAEKCRVSGHDDDTECH CML228 NARFEGQD-NTSSRMEGPAGQTRLNASA---DSGGTENHAVQQTAEKCRVSGHDDDTECH Nc350 NARFEGQD-NTSSRMEGPAGQTRLNASA---DSGGTENHAVQQTAEKCRVSGHDDDTECH *..***** ************* ***.* :****:**:*****:******* ******

Nc358

M162W FHSARGSLQSKSFRASAPLQSKIMSTVDELKGDLSELFSKPADCKPR--PHPHPPRPSKR Nc358 FHSARTSLQSKSFRASAPLQSKIMSTVDELKGDLSELFSKPADCKPR--PHPHPPRPSKR Ms71 FHSARASLQSKSFRASAPLQSKIMSTVDELKGDLSELFSKPADCKPRPHPHPHPPRPSKR B73 FHSARASLQSKSFRASAPLQSKIMSTVDELKGDLSEMFSKPADCKPR--PHPHPPRPSKR Mo17 FHSARASLQSKSFRASAPLQSKIMSTVDELKGDLSEMFSKPADCKPR--PHPHPPRPSKR Tzi8 FHSARTSLQSKSFRASAPLQSKIMSTVDELKGDLSELFSKPADCKPR--PHPHPPRPSKR CML247 FHSARTSLQSKSFRASAPLQSKIMSTVDELKGDLSELFSKPADCKPR--PHPHPPRPSKR CML228 FHSARTSLQSKSFRASAPLQSKIMSTVDELKGDLSELFSKPADCKPR--PHPHPPRPSKR Nc350 FHSARTSLQSKSFRASAPLQSKIMSTVDELKGDLSELFSKPADCKPR--PHPHPPRPSKR ***** ******************************:********** ***********

M162W DGGRTARAAVTSSAPLAAYRPPAEHSGYVSRSRLSRSAQVLPPPRRGLPSRGIAGTGVYP Nc358 DGGRTARAAVTSSAPLAAYRPPAEHSGYVSRSRLSRSAQVLPPPRRGLPSLRYRRHRVYP

A

Non-MutantB73

Lgn-R B73

Lgn-R NIL

Non-MutantNIL

GRMZM2G072892

GRMZM2G04911

GRMZM2G119850

Nor

mal

ized

Fol

d Ex

pres

sion

Non-mutant

EMS mutant

Plan

t Hei

ght (

cm)

Leaf Width (cm)

Lgn-R/+

GRMZM2G075262

B

D

Extra-Large G-Like Proteins

Putative EDR4 Homologs

XP 010233885.1 Brachypodium distachyon

Two Uncharacterized Maize Homologs

XP 012462838.1 Gossypium raimondii

XP 012458772.1 Gossypium raimondii

XP012456236.1 Gossypium raimondii

XP 009122127.1 Brassica rapa

XP 009130874.1 Brassica rapa

NP 196138.1 EDR4 Arabidopsis thaliana

NP 001309830.1 SOL Zea mays

XP 002467911.1 Sorghum bicolor

NP 001143106.1 Zea mays

XP 012698511.1 Setaria italica

XP 003557970.1 Brachypodium distachyon

XP 015629715.1 Oryza sativa Japonica Group

XP 015647717.1 Oryza sativa Japonica Group

XP 004958712.1 Setaria italica

XP 021310292.1 Sorghum bicolor

NP 001345623.1 Zea mays

NP 001142932.1 Zea mays

Uncharacterized Grass Homologs

E

0

1

2

3

4

5

6

7

8

C

0

0.5

1.0

1.5

2.0

2.5

3.0

CML228 Ms71Nc350

Nor

mal

ized

Fol

d Ex

pres

sion

Non-Mutant Lgn-R

**

0

50

100150200

250

300350

0 2 4 6 8 10 12 14

Figure 4. Sequence analysis supports GRMZM2G075262 as Sol (A) Partial alignment of SOL sequence in rescuing and non-rescuing maize inbred lines. The non-canonicalZF motif is in a purple box. Amino acid substitutions that segregate with ability to rescue are in orangeboxes. Indels that segregate with ability to rescue are in red boxes. The second ZF domain is labeled on thecartoon and the site of EMS induced point mutation, Sol-G489E, is marked on the cartoon with a blue box.(B) Normalized fold expression of syntenic genes in the Sol QTL interval according to background. All treat-ments represent at least three biological replicates. (C) qRT-PCR results examining Sol expression inmutant and non-mutant siblings in severe (CML228, Ms71) and rescued (Nc350) backgrounds. Two-tailedt-tests found Sol expression to be statistically higher only in the severe mutant backgrounds (CML228: t (2)= 29.9, p = 0.001; Ms71: t (4) = 2.3, p = 0.05) (B,C) Error bars represent standard error. (D) Plant heightsand leaf widths at maturity in an EMS mutagenized Lgn-R population. The plant that was found to containa point mutation in GRMZM2G075262 is shown in red. (E) SOL protein tree. SOL and EDR4 indicated withred stars. Tree generated with MEGA X using species: Zea mays, Brachypodium distachyon, Sorghumbicolor, Oryza sativa japonica, Setaria italica, Arabidopsis thaliana, Brassica rapa, Gossypium raimondii,Solanum lycoperscum, Populus trichocarpa. Note that the bottom four branches represent collapsed nodes.

Figure 5

Nor

mal

ized

Fol

d Ex

pres

sion

10 30 60 1440 10 30 60 14400

0.5

1.0

1.52.0

2.53.0 Sol Pr4

TreatmentControl

Nor

mal

ized

Fol

d Ex

pres

sion

H

12

45

Nor

mal

ized

Fol

d Ex

pres

sion

7

Pr4

WT B73Flg22

Lgn-R B73Flg22

Lgn-R B73chitin

0.8

1

1.2

1.4

1.6

1.8

2

Sol 1hr PAMP Treatment

0.9

1

1.1

1.2

1.3

1.4

1.5PR4 1hr PAMP Treatment

0.8

1

1.2

1.4

1.6

1.8

2

0.9

1

1.1

1.2

1.3

1.4

1.5PR4 1hr PAMP Treatment

SolSol

Control Treatment

0.8

1.0

1.2

1.4

1.6

1.8

2.0

WT B73Flg22

Lgn-R B73Flg22

Lgn-R B73chitin

Nor

mal

ized

Fol

d Ex

pres

sion

0.91.0

1.11.2

1.3

1.4

1.5*

*

*

**

*

*

*

*

*

Figure 5. Sol exhibits expression and localization differences that are dependent on background and treatment.(A-C) SOL-B-YFP localization shown with Merged images (A), the YFP filter (B), and the DAPI filter (C). (D-F) SOL-M-YFP localization shown with Merged images (D), the YFP filter (E), and the DAPI filter (F). (G) Sol and Pr4 normalized fold expression as determined by RT-qPCR at four different time points throughout chitin exposure. Two-tailed t-tests found Sol expression to be statistically greater in the treatment versus the control at the 10 min (t (5) = 2.59, p = 0.05), 30 min (t (3) = 2.3, p = 0.01), and 60 min (t (5) = 3.4, p = 0.02) timepoints. t-tests found Pr4 induction to be significant at the 30 min (t (3) = 3.2, p = 0.05) and 60 min (t (3) = 3.5, p = 0.04) timepoints. (H) Sol and Pr4 induction at 60-min time points in B73 andLgn-R B73 backgrounds. Two tailed t-tests found significant Sol induction in non-mutant samples treated with flg22 (t (3) =4.5, p = 0.02) and Lgn-R B73 samples treated with chitin showed significant repression of Sol (t (5) = 2.7, p = 0.04). T-testsalso found significant Pr4 induction in non-mutant B73 treated with flg22 (t (4) = 4.3, p = 0.01) and Lgn-R B73 treated withchitin (t (4) = 8.3, p = 0.001) and flg22 (t (3) = 3.4, p = 0.03.). (G,H) Error bars represent standard error and data representsa minimum of three biological replicates.

DAPIMergeSOL-BA B CYFP

YFPMergeSOL-MD E F DAPI

6

3

0

G

Figure 5

min min

Figure 6

Figure 6. Integrated quantitative RNA-seq and phosphoproteomics reveal a possible MAP kinase signaling cascade that induces immune responses in Lgn-R. (A-C) RNA-Seq of Lgn-R and nonmutant siblings revealed hundreds of significantly differentially expressed genes. Here, we show changes in transcript abundance as fold-change in FPKM (fragments per kilobase of transcript per million mapped reads) in Lgn-R compared to wild-type (WT). Among these genes, the most significantly overrepresented categories are genes encoding WRKY transcription factors (A), PATHOGENESIS-RELATED (PR) proteins that are typically induced when plants detect pathogens (B), and receptors, including several families of receptor-like kinases and Nod-like receptors that are involved in sensing and responding to pathogen infection (C). (D) LGN resides at the plasma membrane and signals through its kinase activity to promote leaf development and block a MAP-kinase immunity cascade. In the presence of the Lgn-R muta-tion, leaf development is compromised and the MAPK cascade is activated leading to a pathogen-triggered immunity (PTI) response which can be dampened by cool temperatures or the presence of Sol-M.

D

A B

C

DOI 10.1105/tpc.18.00840; originally published online June 19, 2019;Plant Cell

Steven Paul Briggs, Jacob O Brunkard and Sarah HakeAlyssa A Anderson, Brian St. Aubin, Maria Jazmin Abraham-Juarez, Samuel Leiboff, Zhouxin Shen,

DISEASE RESISTANCE4 and rescues the liguleless narrow maize mutantThe second site modifier, Sympathy for the ligule, encodes a homolog of Arabidopsis ENHANCED

 This information is current as of October 25, 2020

 

Supplemental Data /content/suppl/2019/07/02/tpc.18.00840.DC2.html /content/suppl/2019/06/19/tpc.18.00840.DC1.html

Permissions https://www.copyright.com/ccc/openurl.do?sid=pd_hw1532298X&issn=1532298X&WT.mc_id=pd_hw1532298X

eTOCs http://www.plantcell.org/cgi/alerts/ctmain

Sign up for eTOCs at:

CiteTrack Alerts http://www.plantcell.org/cgi/alerts/ctmain

Sign up for CiteTrack Alerts at:

Subscription Information http://www.aspb.org/publications/subscriptions.cfm

is available at:Plant Physiology and The Plant CellSubscription Information for

ADVANCING THE SCIENCE OF PLANT BIOLOGY © American Society of Plant Biologists