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In plants, host response to pathogenic microbes is driven both by microbial perception and detection of modified-self. The Xanthomonas campestris effector protein AvrAC/XopAC uridylylates the Arabidopsis BIK1 kinase to dampen basal resistance and thereby promotes bacterial virulence. Here we show that PBL2, a paralog of BIK1, is similarly uridylylated by AvrAC. However, in contrast to BIK1, PBL2 uridylylation is specifically required for host recognition of AvrAC to trigger immunity, but not AvrAC virulence. PBL2 thus acts as a decoy and enables AvrAC detection. AvrAC recognition also requires the RKS1 pseudokinase of the ZRK family and the NOD-like receptor ZAR1, which is known to recognize the Pseudomonas syringae effector HopZ1a. ZAR1 forms a stable complex with RKS1, which specifically recruits PBL2 when the latter is uridylylated by AvrAC, triggering ZAR1-mediated immunity. The results illustrate how decoy substrates and pseudokinases can specify and expand the capacity of the plant immune system., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Xanthomonas campestris pv. campestris (Xcc) colonizes the vascular system of Brassicaceae and ultimately causes black rot. In susceptible Arabidopsis plants, XopAC type III effector inhibits by uridylylation positive regulators of the PAMP-triggered immunity such as the receptor-like cytoplasmic kinases (RLCK) BIK1 and PBL1. In the resistant ecotype Col-0, xopAC is a major avirulence gene of Xcc. In this study, we show that both the RLCK interaction domain and the uridylyl transferase domain of XopAC are required for avirulence. Furthermore, xopAC can also confer avirulence to both the vascular pathogen Ralstonia solanacearum and the mesophyll-colonizing pathogen Pseudomonas syringae indicating that xopAC-specified effector-triggered immunity is not specific to the vascular system. In planta, XopAC-YFP fusions are localized at the plasma membrane suggesting that XopAC might interact with membrane-localized proteins. Eight RLCK of subfamily VII predicted to be localized at the plasma membrane and interacting with XopAC in yeast two-hybrid assays have been isolated. Within this subfamily, PBL2 and RIPK RLCK genes but not BIK1 are important for xopAC-specified effector-triggered immunity and Arabidopsis resistance to Xcc.

ABSTRACT The pathogenic bacterium Xanthomonas campestris pv. campestris, the causal agent of black rot of Brassicaceae, manipulates the physiology and the innate immunity of its hosts. Association genetic and reverse-genetic analyses of a world panel of 45 X. campestris pv. campestris strains were used to gain understanding of the genetic basis of the bacterium's pathogenicity to Arabidopsis thaliana. We found that the compositions of the minimal predicted type III secretome varied extensively, with 18 to 28 proteins per strain. There were clear differences in aggressiveness of those X. campestris pv. campestris strains on two Arabidopsis natural accessions. We identified 3 effector genes (xopAC, xopJ5, and xopAL2) and 67 amplified fragment length polymorphism (AFLP) markers that were associated with variations in disease symptoms. The nature and distribution of the AFLP markers remain to be determined, but we observed a low linkage disequilibrium level between predicted effectors and other significant markers, suggesting that additional genetic factors make a meaningful contribution to pathogenicity. Mutagenesis of type III effectors in X. campestris pv. campestris confirmed that xopAC functions as both a virulence and an avirulence gene in Arabidopsis and that xopAM functions as a second avirulence gene on plants of the Col-0 ecotype. However, we did not detect the effect of any other effector in the X. campestris pv. campestris 8004 strain, likely due to other genetic background effects. These results highlight the complex genetic basis of pathogenicity at the pathovar level and encourage us to challenge the agronomical relevance of some virulence determinants identified solely in model strains. IMPORTANCE The identification and understanding of the genetic determinants of bacterial virulence are essential to be able to design efficient protection strategies for infected plants. The recent availability of genomic resources for a limited number of pathogen isolates and host genotypes has strongly biased our research toward genotype-specific approaches. Indeed, these do not consider the natural variation in both pathogens and hosts, so their applied relevance should be challenged. In our study, we exploited the genetic diversity of Xanthomonas campestris pv. campestris, the causal agent of black rot on Brassicaceae (e.g., cabbage), to mine for pathogenicity determinants. This work evidenced the contribution of known and unknown loci to pathogenicity relevant at the pathovar level and identified these virulence determinants as prime targets for breeding resistance to X. campestris pv. campestris in Brassicaceae.

Xanthomonas campestris pathovar campestris causes black rot, a vascular disease on cruciferous plants, including Arabidopsis thaliana. The gene XC1553 from X. campestris pv. campestris strain 8004 encodes a protein containing leucine-rich repeats (LRRs) and appears to be restricted to strains of X. campestris pv. campestris. LRRs are found in a number of type III-secreted effectors in plant and animal pathogens. These prompted us to investigate the role of the XC1553 gene in the interaction between X. campestris pv. campestris and A. thaliana. Translocation assays using the hypersensitive-reaction-inducing domain of X. campestris pv. campestris AvrBs1 as a reporter revealed that XC1553 is a type III effector. Infiltration of Arabidopsis leaf mesophyll with bacterial suspensions showed no differences between the wild-type strain and an XC1553 gene mutant; both strains induced disease symptoms on Kashmir and Col-0 ecotypes. However, a clear difference was observed when bacteria were introduced into the vascular system by piercing the central vein of leaves. In this case, the wild-type strain 8004 caused disease on the Kashmir ecotype, but not on ecotype Col-0; the XC1553 gene mutant became virulent on the Col-0 ecotype and still induced disease on the Kashmir ecotype. Altogether, these data show that the XC1553 gene, which was renamed avrAC(Xcc8004), functions as an avirulence gene whose product seems to be recognized in vascular tissues.

The cruciferous weedArabidopsis thalianaand the causal agent of black rot disease of CrucifersXanthomonas campestrispv.campestris(Xcc) are both model organisms in plant pathology. Their interaction has been studied successfully in the past, but these investigations suffered from high variability. In the present study, we describe an improved Arabidopsis– Xccpathosystem that is based on a wound inoculation procedure. We show that after wound inoculation,Xcccolonizes the vascular system of Arabidopsis leaves and causes typical black rot symptoms in a compatible interaction, while in an incompatible interaction bacterial multiplication is inhibited. The highly synchronous and reproducible symptom expression allowed the development of a disease scoring scheme that enabled us to analyse the effects of mutations in individual genes on plant resistance or on bacterial virulence in a simple and precise manner. This optimized Arabidopsis– Xccpathosystem will be a robust tool for further genetic and post-genomic investigation of fundamental questions in plant pathology. [ABSTRACT FROM AUTHOR]

Xanthomonas campestris pv. campestris (Xcc) colonizes the vascular system of Brassicaceae and ultimately causes black rot. In susceptible Arabidopsis plants, XopAC type III effector inhibits by uridylylation positive regulators of the PAMP-triggered immunity such as the receptor-like cytoplasmic kinases (RLCK) BIK1 and PBL1. In the resistant ecotype Col-0, xopAC is a major avirulence gene of Xcc. In this study, we show that both the RLCK interaction domain and the uridylyl transferase domain of XopAC are required for avirulence. Furthermore, xopAC can also confer avirulence to both the vascular pathogen Ralstonia solanacearum and the mesophyll-colonizing pathogen Pseudomonas syringae indicating that xopAC-specified effector-triggered immunity is not specific to the vascular system. In planta, XopAC-YFP fusions are localized at the plasma membrane suggesting that XopAC might interact with membrane-localized proteins. Eight RLCK of subfamily VII predicted to be localized at the plasma membrane and interacting with XopAC in yeast two-hybrid assays have been isolated. Within this subfamily, PBL2 and RIPK RLCK genes but not BIK1 are important for xopAC-specified effector-triggered immunity and Arabidopsis resistance to Xcc. [ABSTRACT FROM AUTHOR]

Xanthomonas campestris pv. campestris (Xcc) colonizes the vascular system of Brassicaceae and ultimately causes black rot. In susceptible Arabidopsis plants, XopAC type III effector inhibits by uridylylation positive regulators of the PAMP-triggered immunity such as the receptor-like cytoplasmic kinases (RLCK) BIK1 and PBL1. In the resistant ecotype Col-0, xopAC is a major avirulence gene of Xcc. In this study, we show that both the RLCK interaction domain and the uridylyl transferase domain of XopAC are required for avirulence. Furthermore, xopAC can also confer avirulence to both the vascular pathogen Ralstonia solanacearum and the mesophyll-colonizing pathogen Pseudomonas syringae indicating that xopAC-specified effector-triggered immunity is not specific to the vascular system. In planta, XopAC-YFP fusions are localized at the plasma membrane suggesting that XopAC might interact with membrane-localized proteins. Eight RLCK of subfamily VII predicted to be localized at the plasma membrane and interacting with XopAC in yeast two-hybrid assays have been isolated. Within this subfamily, PBL2 and RIPK RLCK genes but not BIK1 are important for xopAC-specified effector-triggered immunity and Arabidopsis resistance to Xcc. [ABSTRACT FROM AUTHOR]