Pathogen/microbe-associated molecular patterns (PAMPs/MAMPs) trigger plant immunity that forms the first line inducible defenses in plants. The regulatory mechanism of MAMP-triggered immunity, however, is poorly understood. Here, we show that Arabidopsis thaliana transcription factors ETHYLENE INSENSITIVE3 (EIN3) and ETHYLENE INSENSITIVE3-LIKE1 (EIL1), previously known to mediate ethylene signaling, also negatively regulate PAMP-triggered immunity. Plants lacking EIN3 and EIL1 display enhanced PAMP defenses and heightened resistance to Pseudomonas syringae bacteria. Conversely, plants overaccumulating EIN3 are compromised in PAMP defenses and exhibit enhanced disease susceptibility to Pseudomonas syringae. Microarray analysis revealed that EIN3 and EIL1 negatively control PAMP response genes. Further analyses indicated that SALICYLIC ACID INDUCTION DEFICIENT2 (SID2), which encodes isochorismate synthase required for pathogen-induced biosynthesis of salicylic acid (SA), is a key target of EIN3 and EIL1. Consistent with this, the ein3-1 eil1-1 double mutant constitutively accumulates SA in the absence of pathogen attack, and a mutation in SID2 restores normal susceptibility in the ein3 eil1 double mutant. EIN3 can specifically bind SID2 promoter sequence in vitro and in vivo. Taken together, our data provide evidence that EIN3/EIL1 directly target SID2 to downregulate PAMP defenses.

Sexual reproduction in flowering plants involves double fertilization, the union of two sperm from pollen with two sex cells in the female embryo sac. Modern plant breeders increasingly seek to circumvent this process to produce doubled haploid individuals, which derive from the chromosome-doubled cells of the haploid gametophyte. Doubled haploid production fixes recombinant haploid genomes in inbred lines, shaving years off the breeding process. Costly, genotype-dependent tissue culture methods are used in many crops, while seed-based in vivo doubled haploid systems are rare in nature and difficult to manage in breeding programmes. The multi-billion-dollar maize hybrid seed business, however, is supported by industrial doubled haploid pipelines using intraspecific crosses to in vivo haploid inducer males derived from Stock 6, first reported in 1959 (ref. 5), followed by colchicine treatment. Despite decades of use, the mode of action remains controversial. Here we establish, through fine mapping, genome sequencing, genetic complementation, and gene editing, that haploid induction in maize (Zea mays) is triggered by a frame-shift mutation in MATRILINEAL (MTL), a pollen-specific phospholipase, and that novel edits in MTL lead to a 6.7% haploid induction rate (the percentage of haploid progeny versus total progeny). Wild-type MTL protein localizes exclusively to sperm cytoplasm, and pollen RNA-sequence profiling identifies a suite of pollen-specific genes overexpressed during haploid induction, some of which may mediate the formation of haploid seed. These findings highlight the importance of male gamete cytoplasmic components to reproductive success and male genome transmittance. Given the conservation of MTL in the cereals, this discovery may enable development of in vivo haploid induction systems to accelerate breeding in crop plants.

SummaryGram-negative bacteria use a variety of virulence factors including phytotoxins, exopolysaccharides, effectors secreted by the type III secretion system, and cell-wall-degrading enzymes to promote parasitism in plants. However, little is known about how these virulence factors alter plant cellular responses to promote disease. In this study, we show that virulent Pseudomonas syringae strains activate the transcription of an Arabidopsis ethylene response factor (ERF) gene, RAP2.6, in a coronatine insensitive 1 (COI1)-dependent manner. A highly sensitive RAP2.6 promoter-®re¯y luciferase (RAP2.6-LUC) reporter line was developed to monitor activities of various bacterial virulence genes. Analyses of P. syringae pv. tomato DC3000 mutants indicated that both type III secretion system and the phytotoxin coronatine are required for RAP2.6 induction. We show that at least ®ve individual type III effectors, avirulence B (AvrB), AvrRpt2, AvrPphB, HopPtoK, and AvrPphE Pto , contributed to RAP2.6 induction. Gene-for-gene recognition was not involved in RAP2.6 induction because plants lacking RPM1 and RPS2 responded normally to AvrB and AvrRpt2 in RAP2.6 expression. Interestingly, the role of coronatine in RAP2.6 induction can be partially substituted by the addition of avrB in DC3000, suggesting that AvrB may mimic coronatine. These results suggest that P. syringae type III effectors and coronatine act by augmenting a COI1-dependent pathway to promote parasitism.

Pathogenic bacterial effectors suppress pathogen-associated molecular pattern (PAMP)-triggered host immunity, thereby promoting parasitism. In the presence of cognate resistance genes, it is proposed that plants detect the virulence activity of bacterial effectors and trigger a defense response, referred to here as effector-triggered immunity (ETI). However, the link between effector virulence and ETI at the molecular level is unknown. Here, we show that the Pseudomonas syringae effector AvrB suppresses PAMP-triggered immunity (PTI) through RAR1, a cochaperone of HSP90 required for ETI. AvrB expressed in plants lacking the cognate resistance gene RPM1 suppresses cell wall defense induced by the flagellar peptide flg22, a well known PAMP, and promotes the growth of nonpathogenic bacteria in a RAR1-dependent manner. rar1 mutants display enhanced cell wall defense in response to flg22, indicating that RAR1 negatively regulates PTI. Furthermore, coimmunoprecipitation experiments indicated that RAR1 and AvrB interact in the plant. The results demonstrate that RAR1 molecularly links PTI, effector virulence, and ETI. The study supports that both pathogen virulence and plant disease resistance have evolved around PTI. Some of the effectors are recognized by host surveillance systems and trigger a strong resistance when their cognate resistance genes are present (2, 4). Often, this so-called ''genefor-gene resistance'' or effector-triggered immunity (ETI; ref . 4) is activated by an indirect interaction between the resistance protein and the cognate effector protein (5). Three proteins, HSP90, RAR1, and SGT1, play an important role in ETI by regulating the stability of NB-LRR resistance proteins (6-11), but they are not known for a role in PTI regulation. It is thought that the plant resistance gene products somehow sense the virulence activity of these effectors, rather than the effectors themselves, which in turn activates resistance. Supporting this hypothesis, several host proteins have been shown to interact with both effector and resistance proteins and are required for ETI (12-16). However, a role of these proteins in effectormediated virulence function remains to be demonstrated.The P. syringae effector protein AvrB enhances virulence on soybean and Arabidopsis plants lacking cognate resistance genes but triggers ETI on soybean and Arabidopsis plants carrying the resistance genes (17). The virulence function of AvrB is expressed as increased bacterial growth in soybean plants and leaf chlorosis in Arabidopsis plants. The virulence and ETI activity of AvrB have the same structural requirements, suggesting that the virulence function and ETI are intimately connected (17, 18). Therefore, host proteins required for AvrB virulence function may provide a molecular link between effector virulence function and ETI.Here we show that AvrB inhibits PTI through RAR1, a HSP90 cochaperone required for disease resistance gene functions. When expressed in plants, AvrB suppresses plant defenses and enhances bacterial growth in a ...

Potentially useful naturally occurring genetic variation is often difficult to identify as the effects of individual genes are subtle and difficult to observe. In this study, a novel genetic technique called MutantAssisted Gene Identification and Characterization is used to identify naturally occurring loci modulating the hypersensitive defense response (HR) in maize. Mutant-Assisted Gene Identification and Characterization facilitates the identification of naturally occurring alleles underlying phenotypic variation from diverse germplasm, using a mutant phenotype as a ''reporter.'' In this study the reporter phenotype was caused by a partially dominant autoactive disease resistance gene, Rp1-D21, which caused HR lesions to form spontaneously all over the plant. Here it is demonstrated that the Rp1-D21 phenotype is profoundly affected by genetic background. By crossing the Rp1-D21 gene into the IBM mapping population, it was possible to map and identify Hrml1 on chromosome 10, a locus responsible for modulating the HR phenotype conferred by Rp1-D21. Other loci with smaller effects were identified on chromosomes 1 and 9. These results demonstrate that Mutant-Assisted Gene Identification and Characterization is a viable approach for identifying naturally occurring useful genetic variation.

Rp1-D21 is a maize auto-active resistance gene conferring a spontaneous hypersensitive response (HR) of variable severity depending on genetic background. We report an association mapping strategy based on the Mutant Assisted Gene Identification and Characterization approach to identify naturally occurring allelic variants associated with phenotypic variation in HR. Each member of a collection of 231 diverse inbred lines of maize constituting a high-resolution association mapping panel were crossed to a parental stock heterozygous for Rp1-D21, and the segregating F 1 generation testcrosses were evaluated for phenotypes associated with lesion severity for 2 years at two locations. A genome-wide scan for associations with HR was conducted with 47,445 SNPs using a linear mixed model that controlled for spurious associations due to population structure. Since the ability to identify candidate genes and the resolution of association mapping are highly influenced by linkage disequilibrium (LD), we examined the extent of genome-wide LD. On average, marker pairs separated by .10 kbp had an r 2 value of ,0.1. Genomic regions surrounding SNPs significantly associated with HR traits were locally saturated with additional SNP markers to establish local LD structure and precisely identify candidate genes. Six significantly associated SNPs at five loci were detected. At each locus, the associated SNP was located within or immediately adjacent to candidate causative genes predicted to play significant roles in the control of programmed cell death and especially in ubiquitin pathway-related processes.T HE hypersensitive response (HR) mechanism is a widespread and important plant defense response. Characterized by a rapid, localized cell death around the point of attempted pathogen penetration, it is a form of programmed cell death and is usually associated with an acute local resistance response and up-regulation of defense response pathways (Coll et al. 2011). HR and associated events are generally initiated by the products of resistance (R) genes, which trigger HR upon the recognition of specific pathogenderived molecules or molecular events (Bent and Mackey 2007). The HR and related responses are generally associated with resistance to biotrophic rather than necrotrophic pathogens. Among the multiple classes of R genes, those that encode proteins possessing a nucleotide-binding site (NBS) and a leucine-rich repeat (LRR) are the predominant class (Bent and Mackey 2007).The Rp1 locus on maize chromosome 10 carries multiple tandemly repeated NBS-LRR paralogs, some of which confer resistance to specific races of maize common rust conferred by the fungus Puccini sorghi (Hulbert 1997). The locus is meiotically unstable due to a high frequency of unequal crossovers between paralogs (Sudupak et al. 1993). In one such case, unequal crossing over followed by intragenic recombination resulted in the formation of the chimeric gene Rp1-D21 (Collins et al. 1999;Smith et al. 2010). In the resulting gene product, the recognition an...

Jasmonates (JAs) are rapidly induced after wounding and act as key regulators for wound induced signaling pathway. However, what perceives the wound signal and how that triggers JA biosynthesis remains poorly understood. To identify components involved in Arabidopsis wound and JA signaling pathway, we screened for mutants with abnormal expression of a luciferase reporter, which is under the control of a wound-responsive promoter of an ethylene response factor (ERF) transcription factor gene, RAP2.6 (Related to APetala 2.6). The rea1 (RAP2.6 expresser in shoot apex) mutant constitutively expressed the RAP2.6-LUC reporter gene in young leaves. Along with the typical JA phenotypes including shorter petioles, loss of apical dominance, accumulation of anthocyanin pigments and constitutive expression of JA response gene, rea1 plants also displayed cell death and accumulated high levels of JA in response to wounding. The phenotype of rea1 mutant is caused by a gain-of-function mutation in the C-terminus of a mechanosensitive ion channel MscS-like 10 (MSL10). MSL10 is localized in the plasma membrane and is expressed predominantly in root tip, shoot apex and vascular tissues. These results suggest that MSL10 is involved in the wound-triggered early signal transduction pathway and possibly in regulating the positive feedback synthesis of JA.

The partially dominant, autoactive maize disease resistance gene Rp1-D21 causes hypersensitive response (HR) lesions to form spontaneously on leaves and stems in the absence of pathogen recognition. The maize nested association mapping (NAM) population consists of 25 200-line subpopulations each derived from a cross between the maize line B73 and one of 25 diverse inbred lines. By crossing a line carrying the Rp1-D21 gene with lines from three of these subpopulations and assessing the F(1) progeny, we were able to map several novel loci that modify the maize HR, using both single-population quantitative trait locus (QTL) and joint analysis of all three populations. Joint analysis detected QTL in greater number and with greater confidence and precision than did single population analysis. In particular, QTL were detected in bins 1.02, 4.04, 9.03, and 10.03. We have previously termed this technique, in which a mutant phenotype is used as a "reporter" for a trait of interest, Mutant-Assisted Gene Identification and Characterization (MAGIC).