the second site modifier, sympathy for ... - the plant cell · 6/13/2019 · 3 arabidopsis...
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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, [email protected] # 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 ([email protected]). 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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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876
WT Lgn-R Mo17
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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
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Wid
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AFigure 2
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Non-mutant
Lgn-RNIL
Lgn-RB73
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0
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Plan
t Hei
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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
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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
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1
1.2
1.4
1.6
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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*
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*
*
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
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Figure 5
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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.
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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
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