The maize heterotrimeric G protein β subunit controls shoot meristem development and immune responses

Significance

Cereal crops, such as maize, provide our major sources of food and feed. Crop productivity has been significantly improved by the selection of favorable architecture and development alleles; however, crops are constantly under attack from pathogens, which severely limits yield due to a defense–growth trade-off. Therefore, identifying key signaling regulators that control both developmental and immune signaling is critical to provide basic knowledge to maximize productivity. This work shows that the maize G protein β subunit regulates both meristem development and immune signaling and suggests that manipulation of this gene has the potential to optimize the trade-off between yield and disease resistance to improve crop yields.

Abstract

Heterotrimeric G proteins are important transducers of receptor signaling, functioning in plants with CLAVATA receptors in controlling shoot meristem size and with pathogen-associated molecular pattern receptors in basal immunity. However, whether specific members of the heterotrimeric complex potentiate cross-talk between development and defense, and the extent to which these functions are conserved across species, have not yet been addressed. Here we used CRISPR/Cas9 to knock out the maize G protein β subunit gene (Gβ) and found that the mutants are lethal, differing from those in Arabidopsis, in which homologous mutants have normal growth and fertility. We show that lethality is caused not by a specific developmental arrest, but by autoimmunity. We used a genetic diversity screen to suppress the lethal Gβ phenotype and also identified a maize Gβ allele with weak autoimmune responses but strong development phenotypes. Using these tools, we show that Gβ controls meristem size in maize, acting epistatically with G protein α subunit gene (Gα), suggesting that Gβ and Gα function in a common signaling complex. Furthermore, we used an association study to show that natural variation in Gβ influences maize kernel row number, an important agronomic trait. Our results demonstrate the dual role of Gβ in immunity and development in a cereal crop and suggest that it functions in cross-talk between these competing signaling networks. Therefore, modification of Gβ has the potential to optimize the trade-off between growth and defense signaling to improve agronomic production.

Shoots are derived from meristems, pools of self-renewing stem cells that initiate new organs from their daughter cells (1). The development of the shoot apical meristem (SAM) is controlled by the CLAVATA (CLV)-WUSCHEL (WUS) feedback signaling pathway (1). This pathway includes a secreted peptide, CLV3; its leucine-rich repeat receptor-like kinase (LRR-RLK), CLV1; and a homeodomain transcription factor, WUS, which promotes CLV gene expression and stem cell fate (2–7). CLV1 binds and perceives the CLV3 peptide, leading to WUS repression (4, 8, 9). A second LRR protein, CLV2, is a receptor-like protein that controls meristem size in parallel to CLV1 (10, 11). The CLV-WUS feedback loop was discovered in the model species Arabidopsis but is conserved widely, including in cereal crops. Through characterization of maize fasciated ear (fea) mutants with enlarged inflorescence meristems (IMs), the THICK TASSEL DWARF1 (TD1), FASCIATED EAR2 (FEA2), and ZmCLAVATA3/EMBRYO SURROUNDING REGION-RELATED7 (ZmCLE7) genes have been identified as orthologs of CLV1, CLV2, and CLV3 respectively (12–16). In addition to the conventional CLV1 receptor, the LRR receptor-like protein FASCIATED EAR3 (FEA3) represses WUS from below and perceives a distinct CLE peptide, ZmFON2-LIKE CLE PROTEIN1 (ZmFCP1) (15). Therefore, distinct CLV receptors perceive small CLE peptides to maintain the balance of meristem proliferation and differentiation. However, the downstream signaling events from these receptors are not well understood.

Heterotrimeric G proteins, consisting of Gα, Gβ, and Gγ subunits, transduce signals downstream of receptors (17). In the standard animal model, a GDP-bound Gα associates with a Gβγ dimer and a 7-pass transmembrane (7-TM) G protein-coupled receptor (GPCR) in its inactive state. On ligand perception, the GPCR promotes GDP release and binding of GTP by Gα, activating the G proteins and promoting interaction with downstream effectors (17). However, G protein signaling in plants appears to be fundamentally different, and whether plants have 7-TM GPCRs remain under debate (18–20). In contrast, emerging evidence suggests that heterotrimeric G proteins in plants interact with single-pass transmembrane receptors (21–24). For example, the maize Gα subunit COMPACT PLANT2 (CT2) interacts with the CLV2 ortholog FEA2 to control shoot meristem development, and ct2 mutants have enlarged SAMs and fasciated ears (21). Similarly, the Arabidopsis Gβ subunit (AGB1) interacts with another CLV-like receptor, RECEPTOR-LIKE PROTEIN KINASE2 (RPK2), to control Arabidopsis SAM development, and Arabidopsis agb1 mutant SAMs are larger (21, 23).

In addition to their developmental functions, heterotrimeric G proteins also positively regulate plant immunity. For example, AGB1 and EXTRA-LARGE GTP-BINDING PROTEIN2 (XLG2), a noncanonical Gα in Arabidopsis, interact with the immune receptor FLAGELLIN SENSITIVE2 (FLS2) as well as with its downstream kinase BOTRYTIS-INDUCED KINASE1 (BIK1), which is stabilized by this interaction (24), and immunity is compromised in xlg and gβ mutants (24, 25). RNAi suppression of the rice Gβ gene RGB1 causes browning of internodes and ectopic cell death in roots, phenotypes associated with immune defects (26, 27). However, the functions of monocot Gβ genes in development have not been dissected, because CRISPR/Cas9-derived rgb1 null mutants die soon after germination (28, 29).

Here we report that CRISPR/Cas9-induced knockouts of maize Gβ (ZmGB1) are seedling lethal, distinct from Arabidopsis but similar to rice. We found that lethality was due to autoimmunity rather than to a developmental arrest. We rescued lethality by introgressing Zmgb1 CRISPR (Zmgb1^CR) mutants into a suppressive genetic background and found that the mutants had larger SAMs and fasciated inflorescences. We also identified a viable allele of ZmGB1 by map-based cloning of a fasciated ear mutant, fea*183, which preferentially alleviated immune phenotypes. Our study dissects the dual functions of Gβ in shoot meristem development and immune responses, suggesting that modulation of G protein signaling has the potential to optimize the trade-off between yield and disease resistance in crop plants.

Results

Knockout of ZmGB1 Using CRISPR/Cas9 Causes Lethality Due to Autoimmunity.

Maize Gα (CT2) and Arabidopsis Gα and Gβ subunits control meristem development (21, 23, 30). However, the role of Gβ in meristem regulation in the grasses remains obscure, because rice Gβ knockouts are lethal, leading to the proposal that it is essential for growth (28, 29). To study the function of maize Gβ, we used CRISPR/Cas9 to generate multiple alleles, including 1-bp and 136-bp deletions with premature stop codons predicted to result in null alleles (Fig. 1A). Homozygous Zmgb1^CR mutants germinated normally but arrested and turned yellow, then brown, and died at an early seedling stage (Fig. 1B).

Fig. 1.

CRISPR/Cas9 knockouts of ZmGB1 led to autoimmune phenotypes. (A) CRISPR/Cas9 editing of ZmGB1 produced different frameshift alleles. White boxes indicate 5′ and 3′ UTRs, black boxes indicate exons, and black lines indicate introns. The positions of guide RNAs are indicated by red arrows. (B) Zmgb1^CR mutants were lethal at the seedling stage. The pictures were taken at 5, 10, and 12 d after seeds were sown in soil. (Upper) WT. (Lower) Zmgb1^CR mutants. (Scale bar: 1 cm.) (C and D) Trypan blue (C) and DAB (D) staining of WT and Zmgb1^CR mutants showed increased staining in the mutants. (E) PR1 and PR5 expression were up-regulated in the Zmgb1^CR mutants, and both 5-d-old and 10-d-old Zmgb1^CR mutants accumulated significantly more salicylic acid (SA) (F). For E and F, P = 0.0001, Student’s t test; n = 3. (G) YFP-SBP-ZmGB1 localizes to membranes in shoot meristems. (Upper) Leaf cells expressing YFP-SBP-ZmGB1 (green), counterstained with FM4-64 (red), both visible as a thin line and overlapped (yellow) around the cell. (Middle) Following plasmolysis, YFP-SBP-ZmGB1 (arrows) remained colocalized with FM4-64. (Lower) YFP-SBP-ZmGB1 expression was found throughout SAM and tassel inflorescence primordia. (Scale bars: 50 μm.)

The necrotic appearance of Zmgb1^CR mutants, along with the known role of AGB1 in Arabidopsis immune responses (24, 25), prompted us to survey immune markers. We first checked for cell death by staining with Trypan blue. Zmgb1^CR mutants were heavily stained compared with wild-type (WT), suggesting that they were undergoing cell death (Fig. 1C). In support of this, 3,3′-diaminobenzidine (DAB) staining showed that H₂O₂, another marker for immune responses, accumulated in the mutants (Fig. 1D). We also checked the expression of 2 immune marker genes, PATHOGENESIS-RELATED PROTEIN1 (PR1) and PR5, and found that both were significantly higher in Zmgb1^CR mutants (Fig. 1E), as were levels of the defense hormone salicylic acid (Fig. 1F). Similar necrotic phenotypes were found in mutants grown in sterile culture, which together with the up-regulation of immune markers suggests that Zmgb1^CR mutants died because of an autoimmune response.

To confirm that the phenotypes were due to mutation of ZmGB1 and not to an off-target effect of CRISPR/Cas9, we made a translational fusion of the ZmGB1 genomic sequence with YELLOW FLUORESCENT PROTEIN (YFP)-STREPTAVIDIN-BINDING PEPTIDE (SBP) at its N terminus, under the control of its native promoter and terminator. This construct was transformed into maize and backcrossed twice to Zmgb1^CR heterozygotes in the B73 background. The YFP-SBP-ZmGB1 transgene was able to complement the lethal phenotypes of Zmgb1^CR mutants (SI Appendix, Table S1). Imaging revealed YFP-SBP-ZmGB1 localization to the plasma membrane (Fig. 1G), as expected (31) and confirmed by colocalization with FM4-64 after plasmolysis (Fig. 1G). Consistent with its anticipated role in shoot development, ZmGB1 was expressed throughout the SAM and IMs (Fig. 1G).

Having confirmed the Zmgb1 phenotypes, we asked why the phenotypes of Arabidopsis Gβ mutants (reduced immune response, but overall normal growth and fertility) are weaker than in maize. To investigate whether this was due to the differences in the Gβ protein, we expressed maize ZmGB1 in Arabidopsis, driven by the native AGB1 promoter. The ZmGB1 transgene fully rescued the developmental and immune defects of agb1 mutants (SI Appendix, Fig. S1), suggesting that Gβ function is conserved between maize and Arabidopsis, and that the contrasting immune phenotypes are not due to differences in the Gβ protein.

Zmgb1 Lethality Can Be Suppressed.

The early lethality of Zmgb1^CR plants precluded us from observing their meristem phenotypes. Autoimmune phenotypes are common for proteins that are “guardees,” protected (or guarded) by RESISTANCE (R) proteins (32). Since R genes are highly polymorphic across accessions, we attempted to suppress Zmgb1^CR autoimmunity by crossing viable heterozygotes to each of the 25 nested association mapping (NAM) maize diversity lines (33) and then screening for suppression in the F2s. Indeed, we found that the lethality of Zmgb1^CR could be partially suppressed after crossing to a tropical maize line, CML103. The suppressed Zmgb1^CR mutants were dwarfed with wider stems, similar to the maize ct2 (Gα) mutants (Fig. 2A) (21), and some of the plants survived to flowering (Fig. 2B).

Fig. 2.

The lethality of Zmgb1^CR mutants was suppressed in the CML103 background. (A) F2 progeny of a cross between Zmgb1^CR heterozygotes and CML103 segregates for lethal and suppressed phenotypes. The pictures are of 7-d-old maize seedlings. (B) The suppressed Zmgb1^CR plants in the CML103 background grew to the adult stage. (C and D) Zmgb1^CR mutants had enlarged SAMs (C), quantified in (D). P = 0.0001, Student’s t test; n = 18 for WT and n = 12 for Zmgb1^CR. (E) Top-down view of WT and Zmgb1^CR ear primordia in the SEM. IMs are shaded in yellow. (Scale bars: 100 μm in C and 1 mm in E.)

Consistent with this growth recovery, the induction of PR1 and PR5 immune marker genes was reduced in the suppressed Zmgb1^CR mutants (SI Appendix, Fig. S2), confirming that autoimmunity was also suppressed. We took advantage of these lethality-suppressed Zmgb1^CR mutants to study the development of their meristems. The mutants had significantly larger SAMs compared with WT sibs (Fig. 2 C and D) and fasciated IMs (Fig. 2E), indicating that ZmGB1 controls both SAM and IM development in maize.

A Newly Identified Fasciated Ear Mutant, fea*183, Encodes a Viable Allele of ZmGB1.

Concurrently, we identified a viable recessive allele of Zmgb1 by map-based cloning of fea*183, a fasciated ear mutant from an ethyl methanesulfonate (EMS)-mutagenesis screen. fea*183 mutants were semidwarf and had shorter, wider leaves with prominent lesions (Fig. 3A). They also had striking inflorescence defects, including fasciated ears and compact tassels (Fig. 3 B and C and SI Appendix, Fig. S3A), reminiscent of ct2 mutants (21). We analyzed developing ear and tassel primordia using scanning electron microscopy (SEM) and found that their IMs were significantly enlarged (Fig. 3B and SI Appendix, Fig. S3B). In addition to IM defects, fea*183 mutants had larger shoot apical meristems (Fig. 3 D and E). The mutants also had obvious cell death and up-regulation of PR genes, suggesting an autoimmune phenotype, albeit much weaker than that of Zmgb1^CR mutants (SI Appendix, Fig. S3 C and D).

Fig. 3.

Characterization and mapping of the fea*183 mutant. (A) fea*183 plants were semidwarf with upright leaves and lesions (Inset, dotted lines). The lesion part was arrowed. (B) SEM images showing that fea*183 mutant ear primordia had enlarged IMs, shaded in yellow. (Scale bars: 500 μm.) (C) Representative mature cobs of WT and fea*183 showing the fasciated ear phenotype. (D) Cleared SAM images of 12-d-old WT and fea*183 mutants. (Scale bars: 100 μm.) (E) fea*183 mutants had larger SAMs. P = 0.008, Student’s t test; n = 14 for WT and n = 12 for fea*183. (F) Positional cloning of fea*183 mutant identified ZmGB1 as the candidate gene. The vertical lines indicate the position of markers used. The numbers of recombinants at each position are listed in red. (G) fea*183 failed to complement Zmgb1^CR in IM development. Shown are SEM images of ear primordia. (Scale bars: 500 μm.) (H) fea*183 failed to complement Zmgb1^CR seedling development. Pictures are of 2-wk-old seedlings.

Bulked segregant analysis and map-based cloning delineated the fea*183 mutation between 257.3 Mb and 258.9 Mb on chromosome 1 (Fig. 3F and SI Appendix, Fig. S4A). Whole-genome sequencing identified a single nonsynonymous mutation within this region, a G-to-A substitution in the fourth exon of ZmGB1, leading to a change in the amino acid 277 from aspartic acid to asparagine in 1 of the WD40 domains (SI Appendix, Fig. S4B). This residue is fully conserved across a wide range of species, including Saccharomyces cerevisiae, Caenorhabditis elegans, Homo sapiens, and Arabidopsis, implying its essential role in Gβ function (SI Appendix, Fig. S4C). We next confirmed that fea*183 encoded an allele of ZmGB1 by crossing with Zmgb1^CR heterozygous plants. In the F1, approximately one-half of the plants had enlarged IMs and dwarfism, similar to fea*183 mutants (Fig. 3 G and H), indicating a failure to complement and demonstrating that FEA*183 encodes the maize Gβ subunit. Thus, we renamed fea*183 as Zmgb1^fea*183.

We next asked how the D²⁷⁷N mutation affects Zmgb1^fea*183 function, by comparing it with human Gβ, HsGB1, and guided by a structure of the human G protein complex (34). The D²⁷⁷ residue in Zmgb1^fea*183 aligned to D²⁵⁴ in HsGB1 (34) (Fig. 4A), which lies at the interface of Gβ and Gγ (Fig. 4B). We thus asked whether this residue is required to form the heterotrimeric complex, using a yeast 3-hybrid (Y3H) experiment (35). We found that unlike the WT protein, the Zmgb1^fea*183 protein could not form a complex with a maize Gγ subunit (ZmRGG2) and Gα/CT2, or with any of the XLG proteins (Fig. 4C), indicating that Zmgb1^fea*183 is unable to form a heterotrimeric complex and suggesting that it is a null allele. Consistent with this idea, we found that the SAM and IM phenotypes of Zmgb1^fea*183 mutants were indistinguishable from Zmgb1^fea*183/null Zmgb1^CR plants (SI Appendix, Fig. S5).

Fig. 4.

Zmgb1^fea*183 failed to form a protein complex with Gα and Gγ subunits. (A) The D277 residue mutated in Zmgb1^fea*183 aligns to D254 in human HsGB1. (B) D254 highlighted in red in HsGB1 is located at the Gβ–Gγ interface. Viewed by PyMoL, with the Gα subunit in orange, the Gβ subunit in cyan, and the Gγ subunit in green. (C) ZmGB1 and the ZmRGG2 Gγ subunit formed complexes with Gα/CT2 or XLGs in a Y3H assay, while Zmgb1^fea*183 did not. ZmGB1 was fused with the BD domain and coexpressed with RGG2 using a pBridge construct (Clontech). Gα/CT2 or individual XLG proteins were fused with the AD domain in the pGADT7 vector. Yeast growth on synthetic complete-Met-Trp-Leu (SC-MLW) medium confirmed transformation and cell viability. Interactions were assayed on SC-Met-Trp-Leu-His (SC-MLWH) medium supplemented with 1 mM 3-AT.

ZmGB1 Functions in the CLAVATA Pathway.

To further decipher the role of ZmGB1, we made double mutants using the Zmgb1^fea*183 allele with other meristem regulatory genes, including fea2, ct2, and fea3 (13, 15, 21), and measured meristem size in segregating populations. The SAMs and ear IMs of Zmgb1;fea2 double mutants were not obviously different from those of the fea2 single mutant, indicating that fea2 is epistatic to Zmgb1 and suggesting that they act in a common pathway (Fig. 5 A–C). Similarly, IMs of Zmgb1;ct2 double mutants were no more fasciated than either single mutant, suggesting that ZmGB1 and CT2/Gα function together in regulating IM development (Fig. 5D). However, vegetative SAMs of Zmgb1;ct2 double mutants were more severely affected than the single mutant, presumably because CT2 acts redundantly with ZmXLGs during vegetative development (35) (Fig. 5 E and F). Finally, Zmgb1;fea3 double mutants had significantly larger SAMs and more strongly fasciated IMs than either single mutant (Fig. 5 G–I), indicating an additive genetic effect and demonstrating that Zmgb1 and fea3 act in different pathways in both SAM and IM regulation, in line with previous observations (36). In summary, our data suggest that ZmGB1 functions together with CT2/Gα in inflorescence development, downstream of the FEA2 CLAVATA receptor.

Fig. 5.

ZmGB1 functions in a CLAVATA pathway. (A) SEM images of WT, Zmgb1, fea2, and Zmgb1;fea2 ear primordia. The double mutants showed similar IMs as the fea2 single mutant. (B) Representative SAM pictures from 16-d-old WT, Zmgb1, fea2, and Zmgb1;fea2 plants. (C) SAM size quantification showed that the SAM size of the Zmgb1;fea2 double mutants was indistinguishable from that of the fea2 single mutants. (D) SEM images of WT, Zmgb1, ct2, and Zmgb1;ct2 ear primordia. The double mutants showed similar IMs as the single mutants. (E) Representative SAM pictures of WT, Zmgb1, ct2, and Zmgb1;ct2 plants. (F) SAM size was significantly larger in the Zmgb1;ct2 double mutant compared with the single mutants. (G) SEM images of WT, Zmgb1, fea3, and Zmgb1;fea3 ear primordia. The IMs were significantly larger in the double mutant compared with the single mutants. (H) Representative SAM pictures of 16-d-old WT, Zmgb1, fea3, and Zmgb1;fea3 plants. (I) SAM size was significantly larger in the Zmgb1;fea3 double mutant than in the single mutants. In C, F, and I, ANOVA analysis was performed with R. P values, mean values, and replicate numbers are indicated in the figures. (Scale bars: 500 μm for A, D, and G; 100 μm for B, E, and H.)

ZmGB1 Associates with Maize Kernel Row Number.

Kernel row number (KRN) is an important agronomic trait that directly contributes to yield (15, 37, 38). Natural or induced variation in FEA2 or FEA3 is associated with KRN, and manipulation of CT2 also enhances KRN (15, 35, 37). Therefore, we asked whether ZmGB1 also associates with this yield trait by conducting a candidate gene association study using a maize association panel of 368 diverse inbred lines (39). Indeed, we found that 5 SNPs in the first and third exons of ZmGB1 significantly associated with maize KRN (Fig. 6A). However, all of the SNPs were synonymous and did not change the ZmGB1 protein sequence, suggesting that the variation in KRN is due to changes in ZmGB1 expression. These 5 KRN-associated SNPs can form 4 kinds of haplotypes among the 368 lines, 2 of which (Hap3 and Hap4) have significantly more kernel rows than the other 2 (Fig. 6B). For example, Hap4 has on average 1.5 and 2.5 more kernel rows compared with Hap2 and Hap1, respectively (Fig. 6B). However, the frequencies of favorable Hap3 and Hap4 in the association panel are only 2.17% and 4.07%, implying that the favorable ZmGB1 alleles have not been selected during maize breeding. Therefore, our results suggest that natural variation in ZmGB1 influences IM size and KRN, with the potential to benefit maize yields.

Fig. 6.

Association analysis of ZmGB1 with KRN. (A) The dots show multiple coding SNPs that associate positively with KRN over multiple environments, along with their best linear unbiased prediction (BLUP) data. A total of 368 diverse inbred lines were used in the association analysis using the MLM + Q model. Shaded diamonds below the gene model show the SNP linkage disequilibrium by pairwise R² values. (B) Haplotype analysis using the 5 KRN-associated SNPs and the KRN (BLUP) of these haplotypes in the association panel. Multiple comparisons P < 0.05. Chr1.s.number refers to the coordinate of maize chromosome 1 based on B73 V2 genome.

Discussion

Heterotrimeric G proteins are important signal transducers that control many biological processes across a wide range of species (17, 40). They also control many important agronomic traits in cereals (21, 28, 35, 41–44), and understanding G protein signaling requires a study of each subunit. Rice Gβ CRISPR null mutants undergo early developmental arrest and death, but the underlying mechanism was unclear (28, 29). Here we show that maize Gβ null alleles are also lethal, and that this is due to autoimmunity, not to specific developmental defects. We suppressed the Gβ lethal phenotype in the CML103 tropical maize genetic background and identified a viable EMS allele, allowing developmental analysis of Gβ meristems. Using the suppressed CRISPR null and the viable Zmgb1^fea*183 alleles, we show that Gβ controls shoot meristem development. Our results suggest that Gβ interacts with different downstream effectors to function independently in immune and development signaling.

An important question is why only monocot Gβ mutants, such as in rice or maize, but not Arabidopsis mutants, develop autoimmunity. Intriguingly, the Arabidopsis Gβ mutant agb1 has a reduced immune response, in contrast to the autoimmune phenotype in rice or maize (24, 25). Expression of maize Gβ fully complemented the immune defects of Arabidopsis agb1 mutants (SI Appendix, Fig. S1), suggesting that Gβ protein function is conserved, and that the contrasting phenotypes are probably due to differences in immune signaling pathways.

Plants have a 2-tiered immune system. First, pathogen-associated molecular pattern (PAMP) receptors recognize conserved microbial elicitors and induce pattern-triggered immunity (PTI) (45–47). To overcome PTI, pathogens have evolved effectors that they secrete into plant cells to interfere with PAMP signaling, and in turn, plants evolved R genes to activate the stronger effector-triggered immunity (ETI), which often results in programmed cell death (48–51). Some R proteins guard native plant proteins, known as “guardees,” that are targeted by pathogen effectors. Thus, mutation of a guardee may mimic the presence of a pathogen and activate the guarding R protein, resulting in an autoimmune phenotype (52). Therefore, it is reasonable to speculate that grass Gβ proteins function as immune guardees. Supporting this hypothesis, Gβ has 7 WD-40 domains and forms a propeller structure, similar to some other effector targets (53).

Our hypothesis explains why the immune phenotypes of Zmgb1^fea*183 mutants are weaker, because presumably this allele accumulates some (albeit mutant) Gβ protein that can still interact with a hypothetical guard R protein but is recognized as abnormal, initiating a partial autoimmune response. R genes are highly polymorphic across accessions, and our results suggest that Gβ is guarded in the monocots rice and maize, but not Arabidopsis (53). To test this hypothesis, further studies are needed to identify the gene(s) responsible for the suppression of Zmgb1 lethality in CML103.

Our genetic analyses suggest that ZmGB1 works in a common pathway with FEA2 and CT2/Gα but independent of FEA3. fea2 was epistatic to both ct2 (Gα) and Zmgb1 in IM fasciation, suggesting that both G protein subunits function together downstream of the FEA2 receptor. However, ct2/Gα and Zmgb1 phenotypes were additive in the SAM, which could be explained by redundancy with the noncanonical Gα proteins, or XLGs, in the SAM (35). However, Zmxlg triple mutants are also lethal (35), preventing us from making higher-order mutants in maize. Identification of a viable genetic background for Gα higher-order mutants would help address this question.

Geneticists and breeders have used quantitative trait locus (QTL) and genome-wide association analyses to identify genes involved in yield traits. Several yield QTL that correspond to heterotrimeric G proteins or CLV-WUS genes have been cloned in rice, maize, and tomato (38, 42, 54–56). For example, FEA2 is a QTL responsible for variation in maize KRN (37), and a rice Gγ gene, GS3, is a QTL for grain length, weight, and thickness (44), while another rice Gγ gene, DEP1, is a QTL for rice grain yield and nitrogen use efficiency (42, 43). These studies indicate that G proteins and other meristem regulators have the potential to benefit yield traits. In this study, we found that ZmGB1 also associated significantly with KRN under multiple environments, suggesting that it also contributes to quantitative variation in KRN. In rice, overexpression of RGB1 enhances tolerance to biotic and abiotic stresses (57, 58), but grain size is reduced (41), suggesting that more subtle modulation of ZmGB1 expression is needed to optimize yield (15, 37, 59).

Improving crop productivity involves selection of favorable architecture and development alleles. Despite these striking innovations, crops are constantly under attack from pathogens. However, turning on defense signaling often causes reductions in growth and yield (60, 61). This defense–growth trade-off results from the intertwining of defense signaling with physiological networks regulating plant fitness (60). Therefore, an understanding of developmental and immune signaling cross-talk is critical to provide basic knowledge to maximize productivity. Our study shows that ZmGB1 is a critical regulator in both meristem development and immunity; therefore, this gene has the potential to optimize defense–development trade-offs to improve agronomic production.

Materials and Methods

The Zmgb1^CR alleles were created using CRISPR/Cas9, and the Zmgb1^fea*183 allele was obtained from an EMS mutagenesis screen using seed stocks provided by Gerald Neuffer. Complete details regarding materials, experimental methods, and data analyses are provided in SI Appendix. All data are contained in the paper and SI Appendix. All of the data and materials will be available on request from the corresponding author.