The Second Site Modifier, Sympathy for the ligule,Encodes a Homolog of Arabidopsis ENHANCED DISEASERESISTANCE4 and Rescues the Liguleless narrowMaize Mutant[OPEN]

Alyssa Anderson,a,1 Brian St. Aubin,a,1,2 María Jazmín Abraham-Juárez,a,3 Samuel Leiboff,a Zhouxin Shen,b

Steve Briggs,b Jacob O. Brunkard,a and Sarah Hakea,4

a Plant Gene Expression Center, U.S. Department of Agriculture-Agricultural Research Service and University of California Berkeley,Albany, California 94710bDivision of Biological Sciences, University of California San Diego, La Jolla, California 92093

ORCID IDs: 0000-0002-8633-9835 (A.A.); 0000-0002-6656-1826 (B.S.A.); 0000-0002-5468-8176 (M.J.A.-J.); 0000-0002-6623-1181(S.L.); 0000-0002-7487-4064 (Z.S.); 0000-0002-7226-8618 (S.B.); 0000-0001-6407-9393 (J.O.B.); 0000-0001-6953-1529 (S.H.)

Liguleless narrow1 encodes a plasma membrane-localized receptor-like kinase required for normal development of maize(Zea mays) leaves, internodes, and inflorescences. The semidominant Lgn-R mutation lacks kinase activity, and phenotypicseverity is dependent on inbred background. We created near isogenic lines and assayed the phenotype in multipleenvironments. Lgn-R plants that carry the B73 version of Sympathy for the ligule (Sol-B) fail to grow under hot conditions, butthose that carry the Mo17 version (Sol-M) survive at hot temperatures and are significantly taller at cool temperatures. Toidentify Sol, we used recombinant mapping and analyzed the Lgn-R phenotype in additional inbred backgrounds. Weidentified amino acid sequence variations in GRMZM2G075262 that segregate with severity of the Lgn-R phenotypes. Thisgene is expressed at high levels in Lgn-R B73, but expression drops to nonmutant levels with one copy of Sol-M. An EMSmutation solidified the identity of SOL as a maize homolog of Arabidopsis (Arabidopsis thaliana) ENHANCED DISEASERESISTANCE4 (EDR4). SOL, like EDR4, is induced in response to pathogen-associated molecular patterns such as flg22.Integrated transcriptomic and phosphoproteomic analyses suggest that Lgn-R plants constitutively activate an immunesignaling cascade that induces temperature-sensitive responses in addition to defects in leaf development. We propose thataspects of the severe Lgn-R developmental phenotype result from constitutive defense induction and that SOL potentiallyfunctions in repressing this response in Mo17 but not B73. Identification of LGN and its interaction with SOL provides insightinto the integration of developmental control and immune responses.

INTRODUCTION

The expressivity of a mutant phenotype is often dependent onother genes. These second site modifiers have been identifiedthrough mutagenesis screens and crosses to different backgrounds.Background-dependent modifiers have been found for develop-mental pathways as diverse as tomato (Solanum lycopersicum)fruit size andDrosophila (Drosophilamelanogaster) bristle number(Gibert et al., 2005; Rodríguez et al., 2013). Maize (Zea mays) isparticularly rich for the identification of modifiers because of thehigh genetic variation among maize inbred lines: to illustrate this

point, thecodingsequencevariationbetween twomaize inbreds isas great as the coding sequence variation between humans andchimpanzees (Pan troglodytes; Buckler et al., 2006), and thenoncoding intergenic space is also remarkably variable betweeninbreds (Brunner et al., 2005). Examples of modifiers in maizeinclude those for seed protein content (Babu et al., 2015), lesionmimic expressivity (Penning et al., 2004), and virescence (Xinget al., 2014). Few modifiers that affect pleiotropic phenotypes,however, have been analyzed at the molecular level.Liguleless narrow-R (R for reference allele) has striking de-

velopmental phenotypes caused by an EMS-induced point mu-tation that eliminates protein kinase activity (Moon et al., 2013). Asa heterozygote in the inbred line B73, Lgn-R plants are short withnarrow leaves and reduced inflorescences. The adult leaves fail toproperlydevelop ligulesandauricles, structures that are locatedatthe blade/sheath boundary in grasses. The ligule keepswater anddebris from entering into the stem where the axillary bud forms(Chaffey, 2000). The auricle allows the blade to tilt back andmaximize exposure to sunlight.The expressivity of the Lgn-R phenotype is background-

dependent. While the phenotype is severe in B73, plant height,leaf width, and inflorescence and ligule development are allrestored to near wild-type levels in Mo17 (Buescher et al., 2014).

1 These authors contributed equally to this work.2 Current address: Department of Plant Biology, Michigan State Univer-sity, 612 Wilson Road, East Lansing, Michigan 48824.3 Current address: CONACYT. Molecular Biology Department, InstitutoPotosino de Investigación Científica y Tecnológica, San Luis Potosí,78216 Mexico.4 Address correspondence to: hake@berkeley.edu.The author responsible for distribution of materials integral to the findingspresented in this article in accordance with the policy described in theInstructions for Authors (www.plantcell.org) is: Sarah Hake (hake@berkeley.edu).[OPEN]Articles can be viewed without a subscription.www.plantcell.org/cgi/doi/10.1105/tpc.18.00840

The Plant Cell, Vol. 31: 1829–1844, August 2019, www.plantcell.org

To investigate thesebackgrounddifferences,wecrossedLgn-R inB73 to the intermatedMo173B73 recombinant inbred lines (IBMlines; Lee et al., 2002) and conducted a quantitative trait locus(QTL) analysis. Sympathy for the ligule (Sol) was identified asa main effect QTL on chromosome 1 (Buescher et al., 2014). Agenotype-by-environment (GxE) effect was discovered by growingthe population in two different environments. Lgn-R B73 mutantsfailed to grow in the hot summers of West Lafayette, Indiana, butsurvived to reproduce in the cooler weather of Albany, California.Phenotypic expressivity that is dependent on background andtemperature is often seen in autoimmune mutants in other plantspecies (van Wersch et al., 2016).

Here, we describe Lgn-R in the near isogenic line (NIL) thatcontains Mo17 at Sol in a predominantly B73 background andidentify the gene for Sol by fine-mapping and expression anal-ysis. Sol encodes a homolog of Arabidopsis (Arabidopsisthaliana) ENHANCED DISEASE RESISTANCE4 (EDR4). EDR4binds to EDR1, a MAP kinase kinase kinase (MAP3K), and lo-calizes it to the site of hyphal penetration pegs (Wu et al., 2015).Like EDR4, Sol is transcriptionally expressed in response totreatment with pathogen-associated molecule patterns(PAMPs). Sol transcript levels are also increased in expressionunder normal growth conditions inLgn-RB73but not inLgn-RNILand nonmutant B73 or Mo17 siblings. Thus, the NIL has bothdevelopmental phenotypes and Sol expression levels that moreclosely resemble its nonmutant siblings.Basedon transcriptomic,proteomic, and phenotypic analyses, we hypothesize that theLgn-R mutation triggers an autoimmune syndrome and that theNIL modifies this response. Our data begin to highlight hypoth-eses toexplain themodifyingbehaviorofSolonLgn-Rand identifyimportant molecular components that contribute to the Lgn-Rphenotype.

RESULTS

The NIL Dampens the Lgn-R Mutant Phenotype

To investigate thedifference inexpressivitybetweenB73andMo17,we created NILs by first crossing Lgn-R to the IBM recombinantinbreds (Buescher et al., 2014) and then backcrossing the leastsevere Lgn-R individuals to B73 for a minimum of four generations.We refer to theMo17Sol locusasSol-Mand theB73 locusasSol-B.Phenotypes were assessed in Lgn-R heterozygotes, and plantswere either Sol-M/Sol-B (NIL) or Sol-B/Sol-B (B73).We grew Lgn-R NIL plants over multiple seasons to determine

the effect on plant architecture in comparison with Lgn-R in theB73 and Mo17 backgrounds (Figures 1A to 1C). Whereas Lgn-RMo17 has nearly the same height and leaf width as the nonmutantMo17 inbred, both height and width are reduced in the NIL, andmost severe in B73 (Figures 1D to 1G; Supplemental Figures 1Aand1B). InAlbany,California, nonmutant plants averaged179.4610.5 cm in height and 106 0.6 cm in leaf width, Lgn-R NIL plantsaveraged 147.4610.2 cm in height and 7.560.9 cm in leafwidth,andLgn-RB73averagedonly102.4622.4cm forplantheight and4.16 0.7 cm for leaf width. Ear development was also restored inLgn-R NIL plants compared with Lgn-R B73 (Figures 1A and 1H).Nonmutant B73 and Mo17 plants as well as Lgn-R Mo17

mutants had morphologically normal ligules and auricles locatedbetweendistinct blade and sheath regions. The ligule inLgn-RNILplants formed across the leaf but the auricle was only partiallyrestored and, as previously described (Moon et al., 2013), Lgn-RB73 plants never developed a complete ligule or auricle in adultleaves (Figures 1B and 1C).To elucidate the dosage effect of Sol-M on Lgn-R in the B73

background, we self-pollinated the Lgn-R NIL and analyzed the

1830 The Plant Cell

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, and Lgn-R B73. Ears are circled in red.(B) Adaxial view of ligules from leaf 6. The Lgn-R B73 ligule is in the red box.(C) Abaxial view of ligules from leaf 6 in same order as in (B). Auricles are marked by red angles.(D) to (G) Plant height and leaf width measurements. Plants that died are represented on graphs with 0 cm height and 0 cm width. The number of dead isshown with n values.(D)Plant heightmeasurements forLgn-R segregating 1:1 inMo17andB73.Aone-wayANOVAwithaTukey’sposthoc test (95%CI) showed thatLgn-RB73is statisticallymore severe thanLgn-RMo17 inAlbany (P<1e-7). InDavis, theproportionofdeadplantsper genotypewas found tobestatistically significantby z test for Lgn-R B73 plants compared with Lgn-R Mo17 (z 5 235.0, P < 0.00001).(E)Plant leaf widthmeasurements for Lgn-R segregating 1:1 inMo17 andB73. A one-way ANOVAwith a Tukey’s posthoc test (95%CI) showed that Lgn-RB73 is statistically more severe than Lgn-R Mo17 in Albany (P < 1e-7).(F) Plant height measurements for Lgn-R segregating 1:1 in NIL (Sol-B/Sol-M) and B73 (Sol-B/Sol-B) backgrounds. A one-way ANOVA with a Tukey’sposthoc test (95% CI) showed significant differences between Lgn-R NIL and Lgn-R B73 plants (P 5 0.02). In Davis, the proportion of dead plants pergenotype was found to be statistically significant by z test for Lgn-R NIL plants compared with Lgn-R B73 (z 5 28.25, P < 0.00001).(G)Plant leafwidthmeasurements for Lgn-R segregating 1:1 inNIL andB73backgrounds. A one-wayANOVAwith aTukey’sposthoc test (95%CI) showedsignificant differences between Lgn-R NIL and Lgn-R B73 plants (P 5 3.5e-6).(H)Percentageofplants thatdevelopears.Az test foundthat theproportionofLgn-RNILplantswithears issignificantlygreater thantheproportionofLgn-RB73plants that form ears (z 5 231.0, P < 0.00001). Error bars represent SE.(I) and (J) Plant height and leaf measurements for a family segregating for Lgn-R and Sol. A one-way ANOVA with a Tukey’s posthoc test (95% CI) foundsignificant differences between heterozygous and homozygous Lgn-R regardless of Sol genotype.(I)Within Lgn-R heterozygotes, the NIL and Sol-M/Sol-M plants have greater plant heights than their B73 siblings (P < 0.007 and P < 0.004), but there arenot significant differences between the NIL and Sol-M/Sol-M individuals. Among the Lgn-R homozygotes, the NIL has statistically shorter heights thanSol-M/Sol-M plants (P < 0.02) and is significantly taller than Sol-B/Sol-B (P < 0.007).(J)Within Lgn-R heterozygotes, the NIL and Sol-M/Sol-M plants have greater leaf widths than Sol-B/Sol-B (P < 2e-4 and P < 2e-6) but are not significantlydifferent from each other. Within Lgn-R homozygotes, we see a clear dosage effect with each copy of Sol-M, with heterozygotes significantly wider thanSol-B/Sol-B plants (P < 0.004) and significantly narrower than Sol-M/Sol-M plants (P < 9e-4).(K) Adaxial view of the ligule region of Lgn-R heterozygotes in order from top to bottom of Sol-M/Sol-M, Sol-M/Sol-B, and Sol-B/Sol-B.

Sympathy for the ligule: A Maize Homolog of AtEDR4 1831

segregating genotypes in the greenhouse. Of the Lgn-R hetero-zygotes, theNILandSol-M/Sol-Mplantshadgreaterplant heightsand leaf widths than their B73 siblings (height and width: P < 2e-4and P < 2e-6), but there were no significant differences betweenthe NIL and Sol-M/Sol-M individuals (Figures 1I and 1J;Supplemental Figure 1C). The ligule itself, however, was nearnormal, with two copies of Sol-M (Figure 1K). Among the Lgn-Rhomozygotes, the Sol-M allele exhibited a clear dosage effect inthat theNILhadstatisticallygreaterheightsandwidths thanSol-B/Sol-B individuals (P < 0.007 and P < 0.004) but had significantlysmaller heights andwidths thanSol-M/Sol-M plants (P < 0.02 andP < 0e-00; Figures 1I and 1J; Supplemental Figure 1C). Thus, onecopy of Sol-M can improve the growth of both Lgn-R hetero-zygotes and homozygotes, but Lgn-R homozygotes retain mul-tiple defects.

Sol Rescues Temperature-Dependent Lgn-R Lethality

A GxE effect was observed when the original Lgn-R 3 IBM lineswere analyzed in West Lafayette, Indiana, and Albany, California(Buescher et al., 2014). To explore the underlying cause, plantswere grown at different temperatures. Lgn-R NIL plants segre-gating for Sol-M were grown in Davis, California, where averagedaytime temperatures during our field seasons were 33.1°Ccompared with that of West Lafayette, Indiana, at 29.0°C andAlbany, California, at 23.2°C (data available at http://www.ncdc.noaa.gov). Nights have similar temperatures in Davis and Albanyand are slightly warmer in West Lafayette.

Lgn-R plants showed significant GxE effects between the Al-bany, California, and Davis, California, fields (Figures 1D to 1G). Aone-way ANOVA with a Tukey’s posthoc test (95% confidenceinterval [CI]) found significant effects from genotype (P 5 2e-16),location (P 5 2e-16), and genotype-by-location interaction (P 51.4e-11) when comparing the Lgn-R B73 and Lgn-R Mo17populations aswell as significant effects fromgenotype (P52.7e-11), location (P 5 0.0019), and genotype-by-location interaction(P5 4e-7) when comparing Lgn-RNIL and Lgn-RB73 siblings. Allpairwise comparisons can be found in Supplemental Figures 1Aand 1B. For example, during the 2015Davis field season, 17 out of19 Lgn-R plants that were homozygous B73 at the Sol locus diedwithin 5 weeks postgermination (Figures 2A and 2B). By contrast,only 1 out of 10 Lgn-R NIL plants died. None of the Lgn-R plantsdied in Albany. The Lgn-R NIL plants that survived in Davis wereshorter and had narrower leaves compared with their Albanycounterparts. Nonmutant plants survived at both locations andwere, in fact, taller in Davis (Figure 2B).

Todeterminewhethergrowthat high temperature is sufficient totrigger Lgn-R lethality, we grew segregating Lgn-R NIL familiesunder two different temperature regimes in otherwise constantgrowth chambers (15–32°C and 11–21°C). The cooler tempera-ture within these ranges was maintained at night and the warmertemperature was used during the day in an attempt to mimic fieldconditions. In the cool cycling growth chamber, the genotypeshad nearly identical phenotypes, whereas Lgn-R NIL plants werenoticeably more severe in the warm cycling chamber and Lgn-RB73 plants were the most strongly affected (Figure 2C). Afterremoval from the warm cycling chamber, nonmutant and Lgn-RNIL plants continued to grow and form reproductive tissues while

Lgn-R B73 plants never recovered (Figure 2D). We also grew thesegregating populations at either a constant 17 or 30°C. Signif-icant reduction in plant height and leaf width was seen across allgenotypes at 30°C comparedwith 17°C, with Lgn-RB73 themoststrongly affected by the 30°C condition. Intriguingly, some of thephenotypic differences seen under normal field conditions wereeliminated in the 17°C growth chamber experiment. Althoughleaves are still narrower in Lgn-R B73 compared with NIL andnonmutant siblings, plant height was not significantly differentbetween any of the genotypes at this temperature and liguledevelopment was restored in Lgn-R B73 plants at 17°C (Figures2E to 2H; Supplemental Figure 1D). In summary, Lgn-R pheno-types are more severe at higher temperatures and rescued atcooler temperatures and in the presence of theMo17 allele ofSol.

Sol Is an Ortholog of EDR4

To identify the Solmodifier, we genotyped plants from Lgn-RNILfamilies and scored the phenotypes (Supplemental Table 1; see“Materials and Methods”). With recombinant mapping techni-ques, we were able to place Sol between IDP1489 at 56,572K bpandbnlg2238 at 55,080Kbpon chromosome1 (Figure 3A). Basedon the idea that syntenic genes aremost likely to be expressed asproteins (Walley et al., 2016), we used theComparative Genomicswebsite to identify four maize genes in the interval that have or-thologs in sorghum (Sorghum bicolor), rice (Oryza sativa), Setariaitalica, and Brachypodium distachyon (Lyons and Freeling, 2008).The syntenic genes included GRMZM2G075262, a gene of un-known function, GRMZM2G072892, a putative LUNG-7 trans-membrane receptor, GRMZM2G049211, a putative vacuolar sortingreceptor precursor, and GRMZM2G119850, a putative receptor-likekinase.To continue investigating this interval, we crossed Lgn-R in B73

to the NAM Founder lines (McMullen et al., 2009) to determinewhichadditional lines rescued theLgn-Rmutationat theSol locus.TwentyF1Lgn-Rhybridpopulationsweregrown in Indiana,whereLgn-R in B73 is severe. One Lgn-R hybrid, Ms71/B73, showeda lethal phenotype.All other hybrids rescued theLgn-Rphenotypeto some extent (Figure 3B), although the rescued phenotype is notnecessarily due to the Sol locus. Eight of the hybrids were suc-cessively backcrossed a minimum of three generations to B73using themost rescued Lgn-R plant in each generation. Given thesevere phenotype of Lgn-R in the Ms71/B73 hybrid, we alsocrossedLgn-R in theMo17background toMs71andbackcrossedto Ms71 at least two generations prior to analysis. To assessenvironment and background effects, we planted these segre-gating families in Davis and Albany during three field seasons.Wedetermined plant height, leaf width, and the genotype of Sol foreach family. We determined that a line rescued Lgn-R if it dis-played increased plant height and leaf width when heterozygousat the Sol locus compared with Sol-B/Sol-B or Sol-Ms71/Sol-Ms71 siblings.Of theNAMfounder linesexamined, fourdidnot rescueLgn-Rat

Sol (Ms71, CML228, CML247, and Nc358) and three lines dis-played rescuedphenotypes (Nc350,Tzi8, andM162W;Figures3Cto 3I; Supplemental Figures 1E to 1K).Of the severe lines, Lgn-R inthe Ms71 inbred behaved similarly to Lgn-R B73. The average plantheightandleafwidthmeasurements forLgn-RSol-Ms71/Sol-Ms71 in

1832 The Plant Cell

Albany were 144.2 6 35.7 and 5.5 6 1.1 cm and in Davis theyaveraged 40.76 43.5 and 3.26 1.8 cm compared with the Lgn-RSol-M/Sol-Ms71measurementsof 156630.6 and8.960.7 cm inAlbany and 112.46 26.4 and 5.16 1.3 cm in Davis (Figure 3C). Aone-way ANOVA followed by a Tukey’s posthoc test (95% CI)found significant effects fromgenotype (P5 8.8e-7), location (P50.0004), and genotype-by-location interaction (P5 0.007) for thefamily. Thesignificance for individual comparisonscanbe found inSupplemental Figure 1E. CML228 was also not capable of res-cuing Lgn-R at Sol. Sol-CML228/Sol-B plants have heights andwidths that are not statistically different fromSol-B/Sol-B relativesat either field, although significant GxE effects were still observedfor individuals within a given genotype (Figure 3D; SupplementalFigure 1F). This same relationship holds true for the inbred lines

CML247andNC358 (Figures 3Eand3F; Supplemental Figures 1Gand 1H).In contrast to the severe lines, three additional lines rescued the

Lgn-R phenotype when heterozygous at Sol. Average plantheights and leaf widths for the Sol-Nc350/Sol-B heterozygoteswere, respectively, in Albany 141.66 14.22 and 6.76 0.7 cm andin Davis 72.0 6 23.5 and 3.5 6 1.0 cm. These averages are allgreater than their Sol-B/Sol-B counterparts, which measured116.4 6 13.4 and 5.3 6 0.7 cm in Albany and 52.5 6 14.7 and1.756 0.5 cm in Davis. A one-way ANOVA followed by a Tukey’sposthoc test (95% CI) found significant effects from genotype(P 5 2e-16), location (P 5 0.0002), and genotype-by-locationinteraction (P 5 1.6e-11) for the family (Figure 3G; Supple-mental Figure 1I). These samemeasurements forSol-Tzi8/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. Dead plants (circled in white) are Lgn-R B73 and flank an Lgn-RNIL plant.(B) Height measurements of individuals segregating for Lgn-R and Sol-M in cool (Albany) versus hot (Davis) climate. Nonmutant siblings include both Solgenotypes. A two-tailed t test showed that Lgn-RNIL plants were significantly different between the two fields [t(49)5 9.1, P5 4e-12], as were Lgn-R B73plants [t(47) 5 15.2, P 5 9e-20].(C) Top, from left to right: wild-type, Lgn-RNIL, and Lgn-RB73plants at 30 dafter planting in the cycling cool growth chamber (11–21°C). At bottom is sameorder in the cycling hot growth chamber (15–32°C).(D)Mature plants 30 d after cycling hot growth chamber treatment from left to right: nonmutant, Lgn-R NIL, and Lgn-R B73. Only Lgn-R B73 plants wereunable to recover from heat treatment.(E) Adaxial view of the ligule of leaf 6 when plants are grown at a constant 17°C.(F) Adaxial view of the ligule of leaf 6 when plants are grown at a constant 30°C.(G) Plant heights and leaf widths for a family grown at a constant 17°C compared with 30°C. A one-way ANOVA revealed significant differences in heightbetween the nonmutant and Lgn-R B73 siblings at 30°C (P < 0.01) but not at 17°C, whereas significant differences in leaf width were seen at bothtemperatures when comparing Lgn-R in B73 with the NIL (P < 1e-4 and P < 5e-3) and nonmutant siblings.(H)Thechange inplant height and leafwidthobservedpergenotype in17°Cconditions comparedwith30°Cconditions. Two-tailed t tests showed thatLgn-RB73 plants weremost significantly altered between the two temperature regimes comparedwith both nonmutant andNIL siblings in terms of both height[t(25)5 4.4, P < 2e-4 and t(13)5 2.5, P < 0.03] andwidth [t(25)5 2.3, P < 0.03 and t(13)5 3.1, P < 8e-3]. Lgn-RNIL lines were not significantly different fromtheir nonmutant siblings.Error bars in (B) and (H) represent SE.

Sympathy for the ligule: A Maize Homolog of AtEDR4 1833

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 crossed once to Lgn-R/1 in B73 and grown in Indiana. Error bars represent SE.(C) to (I) Plant height and leaf width comparisons for families segregating Lgn-R and Sol in our cool (Albany) and hot (Davis) environments. Dead plants arerepresented on graphs with 0 cm height and 0 cm width, and number per genotype (n) is shown.

1834 The Plant Cell

heterozygotes were 158.56 15.4 and 6.56 0.4 cm in Albany andwere significantly increased over their Sol-B/Sol-B relatives,which measured 120.4 6 12.5 and 4.9 6 0.4 cm, with the sametrends in Davis. A one-wayANOVA followed by aTukey’s posthoctest (95% CI) found significant effects from genotype (P 5 1.1e-11), location (P5 6.4e-13), and genotype-by-location interaction(P 5 5.0e-11) for the family (Figure 3H; Supplemental Figure 1J).The third rescuing line,M162W, showed a similar pattern (Figure 3I;Supplemental Figure 1K).

Analysis of single-nucleotide polymorphism (SNP) data fromPanzea (Zhaoet al., 2006) for thegenes in theSolmapping intervalrevealed coding sequence variation in GRMZM2G075262 thatsuggests that this gene is responsible for rescuing Lgn-R phe-notypic defects. Of the four genes, the GRMZM2G049211 alleleswere identical,GRMZM2G072892hadasingle synonymousSNP,GRMZM2G119850 had 4 nonsynonymous SNPs and 18 synon-ymous SNPs, and GRMZM2G075262 had the most differences,with 12 nonsynonymous SNPs, 26 synonymous SNPs, and 8insertions/deletions (indels). The indels were discovered upondirect sequencing of the coding region of this gene in Mo17, B73,and the inbred lines used in crosses. We then asked whethersequence variation correlatedwith a line’s ability to rescue Lgn-R.We found no informative correlations based on the SNPs inGRMZM2G119850. By contrast, 6 of the 12 nonsynonymousSNPs in the GRMZM2G075262 alleles correlated with the abilityof Sol to rescue Lgn-R and 4 of the 7 indels correlated with res-cuing ability. Indeed, lines that could rescue Lgn-R had the Mo17version of these SNPs and indels, while the lines that could notrescue had the B73 versions (Figure 4A; Supplemental Figure 2A).CML228 and CML247 are exceptions to this pattern: they haveMo17-like sequences but do not rescue Lgn-R when heterozygouswith B73 at Sol.

We investigated the expression of the four genes from theSol QTL interval via RT-qPCR. This analysis revealed thatGRMZM2G075262 transcript levels are significantly higher inLgn-R B73 shoot apices than in Lgn-R NIL and nonmutantsiblings.Noneof theothersyntenicgenes in the interval had trendsin expression levels that directly correlated with severity of theLgn-R phenotype (Figure 4B).

To investigate why CML228 and CML247 have GRMZM2G075262sequences similar to Mo17 but fail to rescue, we performed RT-qPCRin populations segregating for Lgn-R in these families. Forcomparison, we usedMs71,which has theB73Sol haplotype andresults in a severe Lgn-R phenotype, and Nc350, which has theMo17 Sol haplotype and rescues Lgn-R (Figure 4A). As hypoth-esized,Sol is induced in Lgn-Rplants in theMs71backgroundbutnot in Lgn-R plants in aSol-Nc350/Sol-B background (Figure 4C).We found that Sol transcripts are increased in lines that are Lgn-Rand CML228/Sol-B. Results with CML247 were inconsistentacross replicates. This result shows that Sol-Nc350, like Sol-M,is capable of reducing Sol-B expression in the heterozygouscondition but that Sol-CML228 is not, despite the similarity insequence toSol-M. Thus, allelic differences in protein sequenceand in regulation of gene expression may both contribute tovariation at Sol.A mutagenesis screen supported our hypothesis that

GRMZM2G075262 is the gene responsible for the effects of theSol locus on the Lgn-R phenotype. Lgn-R plants that had beenbackcrossed three times to Mo17 were mutagenized with EMSand crossed onto B73. The resulting 2000 kernels were planted inthe field and scored for Lgn-R phenotypes. Whereas almost all ofthe 1000 Lgn-R plants showed partial rescue of plant height andleaf width, we identified one plant with a more severe Lgn-Rphenotype that, when sequenced at GRMZM2G075262, con-tained aG489Emissensemutation inSol-M (Figures 4A, blue line,and 4D; Supplemental Figure 2A, blue box). We did not observeanydevelopmentalphenotypesofSol-G489EwithoutLgn-R in thebackground.GRMZM2G075262, henceforth referred to as Sol, encodes

a homolog of Arabidopsis EDR4. We used BLASTp to identifysimilar proteins in representative plant species, including severalgrasses and eudicots, and constructed a phylogenetic tree todescribe the relationshipof theseproteins.SOLandAtEDR4are ina monophyletic clade of closely related proteins, confirming thatthe two are homologs (Figure 4E). AtEDR4 andSOL share 61.36%identity across 42% of the protein, which includes a C-terminalZn ribbon 12 domain and an N-terminal Zn finger-like domain(Supplemental Figure 2B). The maize genome includes another

Figure 3. (continued).

(C) Family segregating at Sol for Mo17 and Ms71 alleles; heterozygotes are significantly rescued compared with homozygotes. A one-way ANOVA witha Tukey’s posthoc test (95%CI) found a significant difference in leaf width in Albany (P5 1.3e-4). A z test performed on the proportion of dead in Davis alsofound significant differences (z 5 211.8, P < 0.00001).(D) Family segregating at Sol for B73 and CML228 alleles; heterozygotes are not statistically different from homozygotes at either location.(E) Family segregating at Sol for B73 and CML247; heterozygotes are not statistically different from homozygotes at either location.(F) Family segregating at Sol for B73 and Nc358; heterozygotes are not statistically different from homozygotes at either location.(G) Family segregating at Sol for B73 and Nc350 alleles; heterozygotes display a significant rescued phenotype at both locations compared with ho-mozygotes.Aone-wayANOVAwithaTukey’sposthoc test (95%CI) foundasignificant difference in leafwidth inAlbany (P50.01). A z test performedon theproportion of dead in Davis also found significant differences (z 5 227.5, P < 0.00001).(H)Family segregatingatSol forB73andTzi8alleles; heterozygotesdisplayasignificant rescuedphenotypeatboth locationscomparedwithhomozygotes.Aone-wayANOVAwithaTukey’sposthoc test (95%CI) foundasignificantdifference in leafwidth (P51.5e-5) andplantheight (P57.3e-4) inAlbany.A z testperformed on the proportion of dead in Davis also found significant differences (z 5 211.8, P < 0.00001).(I) Family segregating at Sol for B73 andM162W. A one-way ANOVAwith a Tukey’s posthoc test (95%CI) found a significant difference in leaf width(P 5 0.01) in Albany. A z test performed on the proportion of dead heterozygotes compared with homozygotes in Davis also found significantdifferences (z 5 214.9, P < 0.00001).

Sympathy for the ligule: A Maize Homolog of AtEDR4 1835

Figure 4. Sequence Analysis Supports GRMZM2G075262 as Sol.

(A) Partial alignment of SOL sequence in rescuing and nonrescuing maize inbred lines. The noncanonical ZF motif is in a purple box. Amino acid (AA)substitutions that segregatewith ability to rescue are in orange boxes. Indels that segregatewith ability to rescue are in red boxes. The secondZFdomain islabeled on the cartoon, and the site of an EMS-induced point mutation, Sol-G489E, is marked on the cartoon with a blue line.(B)Normalized foldexpressionof syntenic genes in theSolQTL interval according tobackground.All treatments represent at least threebiological replicates.(C)RT-qPCRresultsexaminingSolexpression inmutantandnonmutantsiblings insevere (CML228andMs71)andrescued (Nc350)backgrounds.Two-tailedt tests found Sol expression to be statistically higher only in the severe mutant backgrounds [CML228, t(2)5 29.9, P5 0.001; Ms71, t(4)5 2.3, P5 0.05].Error bars in (B) and (C) represent SE.(D) Plant heights and leaf widths at maturity in an EMS-mutagenized Lgn-R population. The plant that was found to contain a point mutation inGRMZM2G075262 is shown in red.(E)SOLprotein tree. SOLandEDR4are indicatedwith red stars. The treewasgeneratedwithMEGAXusing the followingspecies:Zeamays,Brachypodiumdistachyon, Sorghum bicolor, Oryza sativa japonica, Setaria italica, Arabidopsis thaliana, Brassica rapa, Gossypium raimondii, Solanum lycopersicum,Populus trichocarpa. Note that the bottom four branches represent collapsed nodes.

three EDR4-like genes within the same clade as Sol; however,these have no assigned function and have not been previouslyinvestigated. The only EDR4-like protein that has been intensivelystudied is AtEDR4 (Wu et al., 2015).

We also determined the cellular compartment for SOL-M andSOL-B. Using Nicotiana benthamiana for 35S:Sol-B-YFP and35S:Sol-M-YFP transient expression, we determined that SOL-Blocalizes to the cell periphery and nucleus. SOL-M, by contrast,localized only to the cell periphery (Figures 5A to 5F). Althoughthese experiments were performed in N. benthamiana and notmaize, they support the idea that SOL-B and SOL-M havefunctional differences. Our combined data from analysis of Lgn-Rin multiple inbreds, gene expression levels in different geneticbackgrounds, a revertant screen, and cell localization all stronglysupport the identity of Sol as GRMZM2G075262, a maize ho-molog of EDR4.

Sol Expression Is Induced by Elicitors of Innate Immunity

AtEDR4 expression shows induction 1 h after treatment with thebacterial elicitors flg22 andHrpZ (Winter et al., 2007). To test ifSolis also transcriptionally induced in response to pathogen elicitors,wemeasuredSol transcript levels 10, 30, and60minand24haftertreatment with chitin (a fungal elicitor) or water (control). Endo-chitinase PR4 (PR4), known to be induced in maize by chitin andother elicitors (Zhanget al., 2017),wasused toconfirm theefficacyof the treatments. By RT-qPCR, we found that Sol was stronglyincreased inexpressionwithin1hafter treatmentwith chitin inB73(wild-type) leaves (Figure 5G). We also treated B73 with flg22 (abacterial elicitor) and treatedLgn-RB73 tissuewith chitin,flg22, orwater and collected samples at the 60-min time point. We foundthatSol in B73was also induced by flg22 at the 60-min time point,whereas Sol levels were not statistically induced in Lgn-R aftertreatment with chitin or flg22 (Figure 5H), presumably becausemock-treated Lgn-R leaves already showed significant inductionlevels compared with the nonmutant B73 controls. In fact, weobserved a significant reduction in Sol expression at the 60-mintime point in Lgn-R tissue treated with chitin compared with thecontrol. The induction of Sol by elicitors indicates conservation ofthis particular transcriptional response across species, support-ing its identity as a homolog of EDR4.

Lgn-R Putatively Triggers a MAP Kinase Innate ImmunitySignaling Cascade

To narrow our search for pathways affected by the Lgn-R mu-tation, we used a transcriptomic approach to identify differentiallyexpressed genes (DEGs) in Lgn-R versus nonmutant siblings. Wefound 1568 DEGs (false discovery rate [FDR] < 0.05) in Lgn-Rshoot apices compared with nonmutant siblings, of which 1119were induced and 448were repressed. A large number of the Lgn-R DEGs are related to disease resistance, including induction ofgenes that encode receptor-like kinases, WRKY transcriptionfactors, several classicalPATHOGENESIS-RELATEDPROTEINS,and nucleotide binding Leu-rich repeat Nod-Like Receptors(NLRs; Figures 6A to 6C; Supplemental Data Set 1). This tran-scriptional signature suggests that an innate immunity signalingcascade is activated inLgn-Rmutants in the absenceof pathogen

infection.Therefore,wehypothesize that lossofLGNactivitycausesan “autoimmune syndrome” with developmental consequences.To identify possible substrates of Lgn-R, we took a global

phosphoproteomic approach. Threebiological replicates of shootapex tissue were collected from Lgn-R B73 and nonmutant sib-lings.Wedetectedphosphopeptides in proteins encodedby3000differentmaizegenes across all samples. Thirty-fiveproteinswerephosphorylated in all wild-type samples but not in any Lgn-Rsamples (Supplemental Table 2); these could therefore be pep-tides that are uniquely phosphorylated by LGN or downstream ofLGN signaling. There is no clear pattern among these potentialsubstrates, although these proteins include a number of tran-scription factors, chromatin-remodeling factors, proteins involvedin cell wall synthesis, and cell cycle regulators that could impactLgn-R phenotypes.EDR4 is proposed to suppress MAP kinase (MAPK)-mediated

immune responses by controlling the localization of aMAP kinasekinase kinase (MAP3K), called EDR1 (or AtMAP3Kd3), which actsas an upstream repressor of AtMPK3/AtMPK6 activity (Wu et al.,2015). Moreover, AtMPK3/AtMPK6-mediated defense signalingis suppressed at cool temperatures (Cheng et al., 2013), similarto the suppression of Lgn-R phenotypes at cool temperatures.These correlations led us to speculate that Sol could function byinteractingwithan immunity-relatedMAPKsignalingcascade thatis activated in theLgn-Rmutant. To investigate this possibility, wesearched for signs of a differentially phosphorylated signalingcascade in theLgn-Rphosphoproteome (comparedwith thewild-type phosphoproteome), focusing on phosphopeptides identifiedin our data set that have strong similarity to well-characterizedphosphorylation sites on proteins in other plant species. With thistargeted approach,we identifiedaputative signaling cascade thatcould act downstream of LGN-R to promote the expression ofpathogen defense-related genes and disrupt development. Al-though not statistically significant, we did identify putative dif-ferentially phosphorylated sites on homologs of BIR3, BAK1,CDG1, BSU1, SHAGGY, MAP3K (YODA-like), MAP2Ks, MAPKs,and WRKYs that are consistent with activation of this innateimmunity MAPK signaling cascade in Lgn-R but not in nonmutantsiblings (Supplemental Data Set 2). Alignments of the sharedphosphosites in BAK1, BSU1, and SHAGGY-Like BIN2 betweenmaize and Arabidopsis are shown in Supplemental Figures 2C to2E. Another canonical MAPK substrate, MKP1, is also hyper-phosphorylated in Lgn-R, further indicating that LGN regulatesMAPK signaling (Park et al., 2011). Thus, we suggest that LGNcould act as an upstream repressor of a BAK1-SHAGGY-MAPKsignaling network that induces expression of pathogendefense genes.

DISCUSSION

Lgn-R is a semidominant maize mutant with striking leaf defects.The phenotype is severe in B73 and nearly wild type in Mo17.Analysis of aNIL that segregates forMo17at theSol locusshowedthat Sol-M rescues severe aspects of the Lgn-R phenotype. Onecopy of Sol-M partially restores the plant height, leaf width, liguledevelopment, ear development, and provides heat stress survivalof the Lgn-R mutant. We mapped Sol to a region containing fourgenes. Subsequent SNP and expression analyses, as well as

Sympathy for the ligule: A Maize Homolog of AtEDR4 1837

a mutagenesis screen, strongly support the hypothesis thatGRMZM2G075262, a maize homolog of EDR4, is responsible forthe Sol phenotypes. Integrated transcriptomics and phospho-proteomics suggest that the Lgn-Rmutation triggers a pathogendefense transcriptional program in the absence of pathogen

infection.Sol-M, but notSol-B, rescuesmany of the developmentdefects and may act in a feedback loop to suppress immunesystem activation.Lgn-R resembles other mutants with constitutively activated

pathogen defense responses in certain regards. Autoimmunemutants are characterized by dwarfism, upregulation of bioticstress genes, and sensitivity to growth conditions and geneticbackground (van Wersch et al., 2016). Plant growth is compro-mised, presumably because more energy is allocated to defensethan to growth and development (Huot et al., 2014). Although fewmaize autoimmunemutants havebeendescribed (Huet al., 1996),Lgn-R appears unique in its developmental leaf defects, such asthe missing ligule and narrow leaves. Many autoimmune mutantscaused by lesions in NLR genes (or related pathways), includingbonsai, chs3-1, chs2-1, and rpp4 in Arabidopsis and Rp1-D21 inmaize, have severe phenotypes at low temperatures that arerescued at higher temperatures (Hua et al., 2001; Jambunathanet al., 2001; Zhang et al., 2003; Yang and Hua, 2004; Huang et al.,2010; Yang et al., 2010; Negeri et al., 2013). Lgn-R follows anopposite pattern in terms of temperature expressivity because itsphenotypes are most severe at high temperature and rescued atlower temperatures. We hypothesize that this difference in tem-perature sensitivity is due to activation of distinct pathogen de-fense signaling networks: NLR-mediated defenses (sometimescalled effector-triggered immunity) are commonly active at lowtemperatures and suppressed at high temperatures, whereasMAPK-mediated defenses (sometimes called pattern-triggeredimmunity) are commonly active at high temperatures and sup-pressed at low temperatures (Cheng et al., 2013). The extremetemperature sensitivity and phosphoproteome of Lgn-R suggestthat it is a mutation in pattern-triggered immunity. The mecha-nisms by which temperature influences pathogenicity, NLR ac-tivity, and MAPK signaling remain unclear (Hua, 2013; Huot et al.,2017); future studies on the precise molecular functions of LGNunder different temperatures could help illuminate how plantdefenses are impacted by the environment in agricultural crops.Lgn-R developmental defects are only partially rescued by the

NIL; the ligule is still incomplete and leaves are narrow comparedwith Lgn-R in Mo17. In the greenhouse, increasing the copynumber of Sol-M does not statistically change the height or leafwidth of Lgn-R heterozygotes but does restore a normal ligule.Sol-M statistically improves growth of Lgn-R homozygotes ina dosage-dependent manner. One copy increases leaf width andplant height and twocopies further improves their growth, but theyare still clearly mutants. These results demonstrate the impor-tance of at least one functional copy of LGN for complete rescueby Sol-M.When Lgn-Rwas crossed into additional inbred lines, a pattern

of amino acid substitutions and indels in Sol became apparentbetween the rescuing and severe inbred lines. There were,however, two exceptions to this pattern, CML247 and CML228.Further investigation showed that, despite their Mo17-like codingregions, at least one, CML228, had significantly increased mRNAlevels in the Lgn-R background when heterozygous with Sol-B.Promoter differences could help to explain this regulatory dif-ference as well as the failure of these alleles to rescue Lgn-R.In further support of the idea that relative expression levels of

Sol could lead to functional differences, EDR4 and Sol are both

Figure 5. Sol Exhibits Expression and Localization Differences That AreDependent on Background and Treatment.

(A) to (C) SOL-B-YFP localization shownwithmerged images (A), the YFPfilter (B), and the DAPI filter (C).(D) to (F)SOL-M-YFP localization shownwithmerged images (D), the YFPfilter (E), and the DAPI filter (F).(G) Sol and Pr4 normalized fold expression as determined by RT-qPCR atfour different time points throughout chitin exposure. Two-tailed t testsfound Sol expression to be statistically greater in the treatment versus thecontrol at the 10-min [t(5)5 2.59, P5 0.05], 30-min [t(3)5 2.3, P5 0.01],and 60-min [t(5)5 3.4, P5 0.02] time points. Two-tailed t tests found Pr4induction to be significant at the 30-min [t(3) 5 3.2, P 5 0.05] and 60-min[t(3) 5 3.5, P 5 0.04] time points.(H) Sol and Pr4 induction at 60-min time points in B73 and Lgn-R B73backgrounds. Two-tailed t tests found significant Sol induction in non-mutant samples treated with flg22 [t(3) 5 4.5, P 5 0.02], and Lgn-R B73samples treatedwith chitin showed significant repression of Sol [t(5)5 2.7,P 5 0.04]. Two-tailed t tests also found significant Pr4 induction in non-mutant B73 treated with flg22 [t(4)5 4.3, P5 0.01] and Lgn-R B73 treatedwith chitin [t(4) 5 8.3, P 5 0.001] and flg22 [t(3) 5 3.4, P 5 0.03].Error bars in (G)and (H) represent SE, anddata representaminimumof threebiological replicates.

1838 The Plant Cell

Figure 6. Integrated Quantitative RNA-Seq and Phosphoproteomics Reveal a Possible MAPK Signaling Cascade That Induces Immune Responses inLgn-R.

(A) to (C) RNA-Seq of Lgn-R and nonmutant siblings revealed hundreds of significantly DEGs. Here, we show changes in transcript abundance as foldchange in fragments per kilobase of transcript per million mapped reads in Lgn-R compared with the wild type. Among these genes, themost significantly

Sympathy for the ligule: A Maize Homolog of AtEDR4 1839

transiently induced 60min after cells are exposed to receptor-likekinase elicitors such as flg22. This result suggests thatSolmaybea pathogen-response gene and that SOL-Mmay act in a negativefeedback loop to limit constitutive activation of defense-relatedMAPK signaling cascades. EDR4 functions in repressing immuneresponses by relocating EDR1, which has a repressive effect onMAPK signaling cascades, to the plasmamembrane in regions ofhyphal penetration. The EDR4 and EDR1 interaction is dependenton the presence of the first 150 amino acids in EDR4 (Wu et al.,2015). Interestingly, the majority of indels and nonsynonymousmutations between SOL-B and SOL-M are within the first 150amino acids of the protein. This finding implies that amino aciddifferences could also be at the heart of the functional differencesbetween the two versions. Additionally, EDR4 is localized at themembrane (Wu et al., 2015), as are SOL-M and SOL-B whentransiently expressed in N. benthamiana. Nuclear localization ofSOL-B and not SOL-M suggests either protein misregulation,subfunctionalization of SOL-B, or simply heterologous mis-expression of the protein. Nonetheless, our results suggesta number of avenues for further investigation, all of which arepotentially related. For example, the amino acid differences couldlead to different binding partners, which in turn lead to alternatelocalization and expression levels. Experiments to elucidate po-tential binding partners of SOL-M versus SOL-B will help unveilfunctional differences between the two proteins.

We propose that LGN is capable of repressing immuneresponses through upstream phosphorylation of a biotic defensesignal transductioncascade (Figure 6D). LGNmayalso function topromote leaf development through an independent signalingnetwork. The ligule and auricle defects found in Lgn-R are uniqueamong autoimmune mutants and could be caused by a distinctdevelopmental pathway affected in the Lgn-R mutants. Recentstudies have linked MAPK signaling cascades to leaf anglephenotypes in rice (Ningetal., 2011).PerhapsLGNaffectsmultipleMAPK pathways, some leading to autoimmune responses andothers leading to distinct developmental events. Sol is tran-scriptionally downstream of the immune signaling cascadecontrolled by LGN and becomes transcriptionally induced in themutant background. We speculate that SOL-M has a similarfunction to AtEDR4 and is capable of suppressing the immuneresponse. SOL-B is incapable of rescuing the Lgn-R phenotype,perhaps because it cannot suppress this immune response. Al-though it is unclear how temperature influences these pathways,the observed phenotypic patterns that we see are more closelyassociated with MAPK-based immune responses than othertypes of immune signal transduction cascades (Cheng et al.,2013).

The identification of themodifier Sol, with its significant geneticand molecular signatures among the maize inbreds, could guide

future breeding efforts to balance pathogen defense with overallgrowth and yield. Further study of the developmental andimmunity-related roles of LGN should reveal new players in therealm of plant resource allocation when under pathogen attackandunravel thecomplexsignalingnetworkunderlying interactionsbetween development and defense.

METHODS

Genetic Material and Crossing Schemes

Lgn-R heterozygotes in amaize (Zeamays) B73 backgroundwere crossedto the IBM lines (Lee et al., 2002) and NAM founder lines (McMullen et al.,2009) andscored in Indiana (Buescher et al., 2014). The tallestLgn-Rplantswere backcrossed to B73 three to six times for mapping and evaluation. Inearlier generations, the most rescued Lgn-R plant in each population wasblindly used for the cross, but later generations usedgenotyping atSolwiththeprimers IDP1489 (Supplemental Table 1).BecauseLgn-R is very severein an Ms71/B73 hybrid, Lgn-R in a B73 background was first crossed toMo17 two times and then crossed toMs71 aminimumof two times aswell.

EMSmutagenesis was performed on pollen from Lgn-R heterozygotesbackcrossed to Mo17 three times and used to pollinate B73 ears. Ap-proximately 2000 kernels were planted in the field and Lgn-R plants wereobserved.SevereLgn-Rplantswerecrossed toB73,andnonmutantswereself-pollinated to create lines that segregated for Sol-M. Individuals thatwere homozygous for Sol-M were sequenced using GRMZM2G075262primers in Supplemental Table 1.

Field and Phenotypic Measurements

Three weeks after planting, plants were numbered and tissue collected forDNA. Plants were scored at 5 weeks for a phenotypic assessment. Plantheight was taken at maturity. Leaf width was measured at the midpoint ofthe blade on the leaf above the ear node. This leaf was consistently thelongest and widest leaf for the nonmutants and usually leaf 6 countingdown from the top of the plant. When Lgn-R plants did not make ears, theleafmeasuredwas the sixth down from the top. InDavis,manyof theLgn-Rplants died prior to maturity. For one season, Lgn-R B73 plants weremeasured for plant height before they died. The average high temperaturefor the months of June through August in Davis, California, is 33.1°C and23.2°C in Albany, California (data from http://www.ncdc.noaa.gov). TheGPS coordinates for the Albany and Davis, California, fields are, re-spectively, (37.887,2122.300) and (38.535,2121.771).One-wayANOVAsandTukey’sposthoc testswereconducted inR. Inanumberof families, theamount of dead plants in Davis did not allow for adequate statisticalanalysis via ANOVA. In these instances, z statistics were calculated ac-cording to the following formula:

z5 p1-p2ð Þ�

p9 � 1-p9ð Þ � 1n1

11n2

� �� �

where p1 and p2 are the proportions for each genotype (i.e., dead plants ingenotype/total plants in genotype), p9 is the combined proportion (i.e., all

Figure 6. (continued).

overrepresented categories are genes encodingWRKY transcription factors (A), PATHOGENESIS-RELATED (PR) proteins that are typically inducedwhenplantsdetect pathogens (B), and receptors, including several families of receptor-like kinases (RLKs) andNod-like receptors that are involved in sensingandresponding to pathogen infection (C).(D) LGN resides at the plasmamembrane and signals through its kinase activity to promote leaf development and block aMAPK immunity cascade. In thepresence of the Lgn-R mutation, leaf development is compromised and the MAPK cascade is activated, leading to a pathogen-triggered immunity (PTI)response that can be dampened by cool temperatures or the presence of Sol-M.

1840 The Plant Cell

dead plants/total plants analyzed), and n1 and n2 are the number of in-dividuals analyzed per genotype.

Growth Chamber and Greenhouse Conditions

Plantsweregrown in16hof full-spectrum lightof 500mmolm22 s21 and8hof dark. In an attempt to mimic field condition temperatures, treatmentsincluded the intervals of 15 to 32°C and 11 to 21°C, where high and lowtemperatures for each condition were matched to light and dark, re-spectively.A furtherexperimentmaintained theplantsataconstant17°Cora constant 30°C. Watering was maintained sufficiently to maintain soilmoisture and up to 1 cm standing in each tray immediately after watering.Soil humidity probes were used to verify consistent hydration betweentemperature conditions.

The greenhouse conditions vary depending on weather, but typicallylight intensity is 215 mmol m22 s21, temperature ranges from 68 to 78°F,humidity from 44 to 76%, and watering through drip emitters occurs ata rate of 850 mL/d.

Fine-Mapping, Markers, and Numbers of Recombinants

To fine-map Sol, Lgn-R NIL plants were backcrossed to B73 and progenywere grown in Berkeley, California. All plants were measured for leaf widthand plant height. In 2012, markers phi109275 at 51,895,132 and bnlg1811at 70,769,955were used to find 41 recombinants froma total of 627plants.In2013and2014,all plantsweregenotyped regardlessof ligulephenotype.Nonmutant plants that were recombinant at Sol were crossed with Lgn-RB73 to assess the phenotype in the next generation. Recombinants thatwere Lgn-R were crossed to B73 to confirm the phenotype in the nextgeneration. Additional markers were used to refine the positions of re-combinants to the region between the markers bnlg2238 and IDP1489.Primer sequences are given in Supplemental Table 1.

Inbred Sequence Analysis

Theoriginal SNPdata set used to analyze the four syntenic genes in theSolQTL interval was the Maize HapMapV2 data (Hufford et al., 2012) found atPanzea.org (Zhao et al., 2006). Using the Genotype Search Tool, SNPswere identified fromall relevant inbred lines in the interval onchromosome1spanning the coding region of all interval genes. B73 RefGen_v3 mappinglocations were used. SNPs present in intronic sequences were ignored.The coding region of GRMZM2G07562 was also directly sequenced in allstudied inbred lines using primers described in Supplemental Table 1.

Phylogenetic Analysis

Sequences were curated via a BLASTp search against the nonredundantprotein sequence collection using the maize SOL sequence. All non-redundant matches with an e value # 0.001 and a minimum of 15%coveragewere selected, downloaded, and aligned usingMUSCLE (Edgar,2004). Sequencesweremanually curated to remove redundant sequencessubmitted with nonredundant names and any obvious protein frag-ments that were less than 100 amino acids in length. This alignment(Supplemental Data Set 3) was fed intoMEGAX (Kumar et al., 2018), whichwe used to compute a maximum likelihood tree with 75 bootstrappingreplicates. A Jones-Taylor-Thornton model and Nearest Neighbor In-terchange ML Heuristic were used while assuming uniform evolutionaryrates between sites.

A specialized function of the BLASTp suite was used to calculatepercentage identity for a SOL and AtEDR4 alignment. Both sequenceswereentered individually using the two-sequenceoption inBLASTp.Thesesequences were then directly aligned and compared using the BLASTpsoftware (Schäffer et al., 2001).

PAMP Treatment and RT-qPCR Analysis

Leaf 4 tissue of 4-week-old plants was collected, and PAMP or mocktreatments were applied according to the method previously described(Zhang et al., 2017). The PAMPs used include flg22 (GenScript) and hexa-N-acetylchitohexaose (Cayman Chemical). Leaf tissue was taken from atleast three separate plants per genotypeper treatment per timepoint. TotalRNA was extracted from these tissues using Trizol Reagent (Invitrogen)according to the manufacturer’s specifications, and cDNA was generatedusing the Superscript III Kits with OligoDt (Thermo Fisher Scientific) fol-lowing the manufacturer’s recommendations. Two-step RT-qPCR (98°Cfor 0.2 s, 55°C for 0.5 s) was conducted on a Bio-Rad CFX96 Real-TimeSystem using primers for the housekeeping gene Gapdh to normalize theexpression of our target genes: Sol and Pr4. All primers are listed inSupplemental Table 1. Expression levels of the analyzed genes werecalculated according to the equation E 5 Peff (2DCt), where Peff is theprimer set efficiency calculated using LinRegPCR (Ramakers et al., 2003)andDCt was calculated by subtracting the cycle threshold (Ct) value of thehousekeeping gene from theCt values of the gene analyzed. Fold changeswere calculated between the ratios of the expression levels of PAMP-treated andmock samples, and expression levelswere calculated for threebiological replicates.

RNA-Seq Material

Seeds of Lgn-R/IBM 3 B73 (BC5) were sown in large pots in the green-house, and 120 plants were genotyped for Sol. Plants that were obviouslyLgn-R were noted, and others were genotyped for Lgn-R, resulting in231/1 Sol-B/Sol-B, 151/1 Sol-M/Sol-B, 24 Lgn-R/1 Sol-B/Sol-B, and24 Lgn-R/1 Sol-M/Sol-B. Shoot apex tissue was harvested 4 weeks afterplanting. To be consistent between genotypes, we made two biologicalreplicateswithsevensamples fromeachgenotype. TheScriptSeqv2RNA-Seq library preparation kit was used in accordancewith themanufacturer’sguidelines for sequencingwith Illumina Hiseq 2000 as SR50.We assessedraw read quality and checked for adapter contamination using fastqc(http://www.bioinformatics.babraham.ac.uk/projects/fastqc) and multiqc(Ewels et al., 2016). Filtered reads were aligned to the maize B73 genomeAGPv3.30 usingHISAT2 (Kim et al., 2015) and assigned to AGPv3.30 genemodels using HTSeq-counts (Anders et al., 2015), using Cyverse Atmo-sphere cyberinfrastructure (Merchant et al., 2016). Counted reads weretested for differential expression with edgeR using a GLM approach toaccount for batch effects between repeated experiments and an FDRsignificance threshold of 0.05 (McCarthy et al., 2012) and differentiallyexpressed fold changes used for additional plots. Gene functional cate-gories were assessed by a parametric test of gene enrichment with Ag-riGOv2 (Tian et al., 2017). For the RNA-seq analysis, we combined the twoLgn-R data sets (Sol-B/Sol-M and Sol-B/Sol-B) and combined the twononmutant data sets to increase the number of replicates in the analysis.

Phosphoproteome

Each pool had ;9 g of shoot tissue isolated from 3-week-old plantssegregating for Lgn-R in the B73 background. Two grams of frozen tissuewas ground in liquid nitrogen by a mortar/pestle for 15 min to fine powderand then transferred to a 50-mL conical tube. Proteins were precipitatedand washed by 50 mL of 220°C acetone three times, then by 50 mL of220°C methanol with 0.2 mM Na3VO4 three times. Protein pellets werealiquoted into four 2-mL Eppendorf tubes per sample and dried ina SpeedVac at 4°C.

Ground tissue powders were suspended in extraction buffer [8 M urea,100 mM Tris, 5 mM Tris(2-carboxyethyl)phosphine, and phosphatase in-hibitors, pH 7]. Proteins were precipitated by adding 4 volumes of coldacetone and incubated at 4°C for 2 h. Sampleswere centrifuged at 4,000g,4°C, for 5 min. Supernatant was removed and discarded. Proteins were

Sympathy for the ligule: A Maize Homolog of AtEDR4 1841

resuspended in urea extraction buffer and precipitated by cold acetone.Proteinpelletswerewashedbycoldmethanolwith0.2mMNa3VO4 tofurtherremove nonprotein contaminants. Protein pellets were suspended in ex-tractionbuffer [8Murea,100mMTris, 5mMTris(2-carboxyethyl)phosphine,and phosphatase inhibitors, pH 7]. Proteins were first digested with Lys-C(Wako Chemicals, 125-05061) at 37°C for 15 min. Protein solution wasdiluted eight times to 1 M urea with 100 mM Tris and digested with trypsin(Roche, 03708969001) for 4 h. Reduced Cys residues were alkylated byadding 10mM iodoacetamide and incubating at 37°C in thedark for 30min.

PhosphopeptideenrichmentwasperformedusingCeO2affinity capture.One percent colloidal CeO2 (Sigma-Aldrich, 289744) was added to theacidifiedpeptidesolution (3%[w/v] trifluoroaceticacid,CeO2:peptide [w/w]51:10). After brief vortexing, CeO2 with captured phosphopeptides wasspun down at 1,000g for 1 min. Supernatant was removed, and the CeO2

pellet was washed with 1 mL of 1% trifluoroacetic acid. Phosphopeptideswereelutedbyaddingelutingbuffer [200mM(NH4)2HPO4,2MNH3$H2O,and10 mM EDTA, pH 9.5, same volume as the added 1% colloidal CeO2] andvortexing briefly. CeO2 was precipitated by adding 10% formic acid with100 mM citric acid (same volume as the added 1% colloidal CeO2) to a finalpH of 3. Samples were centrifuged at 16,100g for 1 min. Supernatantcontaining phosphopeptides was removed for mass spectrometry analysis.

An Agilent 1100 HPLC system was used to deliver a flow rate of 600 nLmin21 to a custom three-phase capillary chromatography column througha splitter. Column phases are a 30-cm-long reverse phase (RP1; 5 mmZorbax SB-C18, Agilent), 5-cm-long strong cation exchange (SCX; 5 mmPolySulfoethyl, PolyLC), and20-cm-long reversephase2 (RP2; 2.5mmBEHC18, Waters), with the electrospray tip of the fused silica tubing pulled toa sharp tip (i.d. < 1 mm). Peptide mixtures were loaded onto RP1, and thethree sections were joined and mounted on a custom electrospray adapterfor online nested elutions. Peptides were eluted from RP1 section to SCXsection using a 0 to 80% acetonitrile gradient for 60 min, and then werefractionated by the SCX column section using a series of 18-step saltgradients of ammonium acetate over 20 min, followed by high-resolutionreverse phase separation on the RP2 section of the column using an ace-tonitrilegradientof0%to80%for120min.One twodimensional-nano-liquidchromatography-tandem mass spectrometry (LC-MS/MS) required 46 h.

Spectra were acquired on LTQ Velos linear ion trap tandem massspectrometers (Thermo Electron) employing automated, data-dependentacquisition.Themassspectrometerwasoperated inpositive ionmodewitha source temperature of 325°C and a spray voltage of 3000 V. The max-imum ion injection time is 50 ms for MS and 100 ms for MS/MS. As a finalfractionation step, gas phase separation in the ion trap was employed toseparate thepeptides into threemass classes prior to scanning; the fullMSscan range was divided into three smaller scan ranges (400–750,750–1000, and 1000–1800 D) to improve dynamic range. Each MS scanwas followedby fiveMS/MSscansof themost intense ions from theparentMSscan.Adynamic exclusion of 1minwasused to improve theduty cycle.

Raw data were extracted and searched using Spectrum Mill vB.06(Agilent Technologies). MS/MS spectra with a sequence tag length of 1 orless were considered to be poor spectra and were discarded. Theremaining MS/MS spectra were searched against the maize B73RefGen_v2 5b Filtered Gene Set downloaded from www.maizesequence.org. The enzyme parameter was limited to full tryptic peptides witha maximum miscleavage of 1. All other search parameters were set toSpectrumMill’s default settings (carbamidomethylation of Cys,62.5 D forprecursor ions, 60.7 D for fragment ions, and a minimum matched peakintensity of 50%). Ox-Met, n-term pyro-Gln, and phosphorylation on Ser,Thr, or Tyr were defined as variable modifications for phosphoproteomedata. A maximum of two modifications per peptide was used. A 1:1concatenated forward-reverse database was constructed to calculate theFDR. The tryptic peptides in the reverse database were compared with theforwarddatabase andwere shuffled if theymatched to any tryptic peptidesfrom the forward database. Common contaminants such as trypsin and

keratin were included in the protein database. There are 127,108 proteinsequences in theproteindatabase.Peptidecutoff scoresweredynamicallyassigned to each data set to maintain the FDR < 0.1% at the peptide level.Phosphorylation sites were localized to a particular amino acid withina peptide using the variable modification localization score in Agilent’sSpectrum Mill software. Proteins that share common peptides weregrouped using principles of parsimony to address the protein databaseredundancy issue. Thus, proteins within the same group shared the sameset or subset of unique peptides. Protein abundance and phosphorylationlevels were quantified via spectral counting. MS runs (replicates) werenormalized so that the total number of spectral counts was equal for eachrun. Spectral counts from technical replicates, when present, were thenaveraged to get the spectral counts for each biological replicate at theprotein level.

Transient Expression in Nicotiana benthamiana

To create 35S:Sol-B-YFP and 35S:Sol-M-YFP, the full-length Sol codingsequence was amplified from cDNA of B73 andMo17 shoots with specificprimers (Supplemental Table 1), cloned into pENTR D, and then re-combined into pEarleyGate 101 (Earley et al., 2006) using the Gatewaytechnology, according to the manufacturer’s instructions (Invitrogen). Theconstruct was transformed into Agrobacterium tumefaciens strain GV3101.Transient transformation of N. benthamiana was performed as described(BolducandHake, 2009). Forty-eight hours after agroinfiltration, leaveswereobserved by LSM710 confocal microscopy (Zeiss) with 470-nm excitationand 535-nm emission filters for YFP and 360-nm excitation and 488-nmemission filters for 49,6-diamidino-2-phenylindole (DAPI) signal. This ex-periment was repeated three times.

Accession Numbers

Sequence data from this work can be found under the following accessionnumbers: Lgn, GRMZM2G134382; Sol, GRMZM2G075262; AtEDR4,AT5G05190; EcPR4,GRMZM2G129189; AtEDR1, AT1G08720; ZmBAK1-like, GRMZM2G115420; ZmBSU1-like, GRMZM2G028700; ZmBIN2-like,GRMZM2G043350; ZmMKP1-like, GRMZM2G005350; RNA-seq data,Bioproject ID PRJNA54785.

Supplemental Data

Supplemental Figure 1. Ninety-five percent CIs for all tukey’s posthoctests, statistical support for figures 1 to 3.

Supplemental Figure 2. Expanded alignments of maize and arabi-dopsis proteins, supporting figures 4 and 6.

Supplemental Table 1. Primers used in this study.

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

Supplemental Data Set 1. DEGs from RNA-Seq.

Supplemental Data Set 2. Phosphopeptides supporting a mAPKsignaling cascade in Lgn-R.

Supplemental Data Set 3. Text file of the alignment used for thephylogenetic analysis shown in figure 4E.

ACKNOWLEDGMENTS

We thank the many students who helped in our summer field seasons,including Yadanar Htike, Sarah Vernallis, Melea Emunah, Nick Stivers,Ashley Noriega, Jamie Jeffries, Haley Kodak, and Alicia Ljungdahl. Thanksto BriannaHaining for sequencing EMSmutations, DeWoods at theUSDA

1842 The Plant Cell

microscopy facility, greenhouse staff, and University of California Davisand University of California Berkeley field staff. Support for this work wasprovided by the National Science Foundation (NSF) Division of IntegrativeOrganismal Systems (IOS-1238202 to A.A. and M.J.A.-J.), by the U.S.Department of Agriculture (CRIS 5335-21000-013-00D to S.H.), by NSF(IOS-1612268 toS.L. and IOS 1546899 toS.B. andZ.S.), and byNIHOfficeof the Director (DP5OD023072 to J.O.B.).

AUTHOR CONTRIBUTIONS

S.H. and A.A. designed the research and wrote the article. A.A. and B.S.A.executed theexperiments.M.J.A.-J. did the localization inN.benthamiana.Z.S. and S.B. carried out the phosphoproteome analysis. J.O.B. and S.L.helped analyze the data.

Received November 6, 2018; revised May 14, 2019; accepted June 13,2019; published June 19, 2019.

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1844 The Plant Cell

DOI 10.1105/tpc.18.00840; originally published online June 19, 2019; 2019;31;1829-1844Plant Cell

Briggs, Jacob O. Brunkard and Sarah HakeAlyssa Anderson, Brian St. Aubin, María Jazmín Abraham-Juárez, Samuel Leiboff, Zhouxin Shen, Steve

Maize MutantLiguleless narrowENHANCED DISEASE RESISTANCE4 and Rescues the Encodes a Homolog of ArabidopsisSympathy for the ligule,The Second Site Modifier,

 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

References /content/31/8/1829.full.html#ref-list-1

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