Pathogens generate and secrete effector proteins to the host plant cells during pathogenesis to promote virulence and colonization. If the plant carries resistance (R) proteins that recognize pathogen effectors, effector-triggered immunity (ETI) is activated, resulting in a robust immune response and hypersensitive response (HR). The bipartite effector AvrRps4 from Pseudomonas syringae pv. pisi has been well studied in terms of avirulence function. In planta, AvrRps4 is processed into two parts. The C-terminal fragment of AvrRps4 (AvrRps4C) induces HR in turnip and is recognized by the paired resistance proteins AtRRS1/AtRPS4 in Arabidopsis. Here, we show that AvrRps4C targets a group of Arabidopsis WRKY, including WRKY46, WRKY53, WRKY54, and WRKY70, to induce its virulence function. Indeed, AvrRps4C suppresses the general binding and transcriptional activities of immune-positive regulator WRKY54 and WRKY54-mediated resistance. AvrRps4C interferes with WRKY54\'s binding activity to target gene SARD1 in vitro, suggesting WRKY54 is sequestered from the SARD1 promoter by AvrRps4C. Through the interaction of AvrRps4C with four WRKYs, AvrRps4 enhances the formation of homo-/heterotypic complexes of four WRKYs and sequesters them in the cytoplasm, thus inhibiting their function in plant immunity. Together, our results provide a detailed virulence mechanism of AvrRps4 through its C-terminus.

Arabidopsis thaliana WRKY proteins are potential targets of pathogen-secreted effectors. RESISTANT TO RALSTONIA SOLANACEARUM 1 (RRS1; AtWRKY52) is a well-studied Arabidopsis nucleotide-binding and leucine-rich repeat (NLR) immune receptor carrying a C-terminal WRKY domain that functions as an integrated decoy. RRS1-R recognizes the effectors AvrRps4 from Pseudomonas syringae pv. pisi and PopP2 from Ralstonia pseudosolanacearum by direct interaction through its WRKY domain. AvrRps4 and PopP2 were previously shown to interact with several AtWRKYs. However, how these effectors selectively interact with their virulence targets remains unknown. Here, we show that several members of subgroup IIIb of the AtWRKY family are targeted by AvrRps4 and PopP2. We demonstrate that several AtWRKYs induce cell death when transiently expressed in Nicotiana benthamiana, indicating the activation of immune responses. AtWRKY54 was the only cell death-inducing AtWRKY that interacted with both AvrRps4 and PopP2. We found that AvrRps4 and PopP2 specifically suppress AtWRKY54-induced cell death. We also demonstrate that the amino acid residues required for the avirulence function of AvrRps4 and PopP2 are critical for suppressing AtWRKY54-induced cell death. AtWRKY54 residues predicted to form a binding interface with AvrRps4 were predominantly located in the DNA binding domain and necessary for inducing cell death. Notably, one AtWRKY54 residue, E164, contributes to affinity with AvrRps4 and is exclusively present among subgroup IIIb AtWRKYs, yet is located outside of the DNA-binding domain. Surprisingly, AtWRKY54 mutated at E164 evaded AvrRps4-mediated cell death suppression. Taking our observations together, we propose that AvrRp4 and PopP2 specifically target AtWRKY54 to suppress plant immune responses.

During pathogenesis, effector proteins are secreted from the pathogen to the host plant to provide virulence activity for invasion of the host. However, once the host plant recognizes one of the delivered effectors, effector-triggered immunity activates a robust immune and hypersensitive response (HR). In planta, the effector AvrRps4 is processed into the N-terminus (AvrRps4N ) and the C-terminus (AvrRps4C ). AvrRps4C is sufficient to trigger HR in turnip and activate AtRRS1/AtRPS4-mediated immunity in Arabidopsis; on the other hand, AvrRps4N induces HR in lettuce. Furthermore, AvrRps4N -mediated HR requires a conserved arginine at position 112 (R112), which is also important for full-length AvrRps4 (AvrRps4F ) processing. Here, we show that effector processing and effector recognition in lettuce are uncoupled for the AvrRps4 family. In addition, we compared effector recognition by lettuce of AvrRps4 and its homologues, HopK1 and XopO. Interestingly, unlike for AvrRps4 and HopK1, mutation of the conserved R111 in XopO by itself was insufficient to abolish recognition. The combination of amino acid substitutions arginine 111 to leucine with glutamate 114 to lysine abolished the XopO-mediated HR, suggesting that AvrRps4 family members have distinct structural requirements for perception by lettuce. Together, our results provide an insight into the processing and recognition of AvrRps4 and its homologues.

Pathogens utilize a repertoire of effectors to facilitate pathogenesis, but when the host recognizes one of them, it causes effector-triggered immunity. The Pseudomonas type III effector AvrRps4 is a bipartite effector that is processed in planta into a functional 133-amino acid N-terminus (AvrRps4-N) and 88-amino acid C-terminus (AvrRps4-C). Previous studies found AvrRps4-C to be sufficient to trigger the hypersensitive response (HR) in turnip. In contrast, our recent work found that AvrRps4-N but not AvrRps4-C triggered HR in lettuce, whereas both were required for resistance induction in Arabidopsis. Here, we initially compared AvrRps4 recognition by turnip and lettuce using transient expression. By serial truncation, we identified the central conserved region consisting of 37 amino acids as essential for AvrRps4-N recognition, whereas the putative type III secretion signal peptide or the C-terminal 13 amino acids were dispensable. Surprisingly, the conserved arginine at position 112 (R112) that is required for full-length AvrRps4 processing is also required for the recognition of AvrRps4-N by lettuce. Mutating R112 to hydrophobic leucine or negatively charged glutamate abolished the HR-inducing capacity of AvrRps4-N, while a positively charged lysine at this position resulted in a slow and weak HR. Together, our results suggest an AvrRps4-N recognition-specific role of R112 in lettuce.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Inducible expression of a pathogen effector has been proven to be a powerful strategy for dissecting its virulence and avirulence functions. However, leaky expression of some effector proteins can cause drastic physiological changes, such as growth retardation, accelerated senescence, and sterility. Unfortunately, leaky expression from current inducible vectors is unavoidable. To overcome these problems, a highly efficient Arabidopsis transformation protocol is described here, which allows the generation of hundreds to over a thousand T1 plants for selecting appropriate lines. In addition, since transgenic silencing is frequently observed, a principle for screening stable transgenic plants is also introduced.

In plant immunity, pathogen-activated intracellular nucleotide binding/leucine rich repeat (NLR) receptors mobilize disease resistance pathways, but the downstream signaling mechanisms remain obscure. Enhanced disease susceptibility 1 (EDS1) controls transcriptional reprogramming in resistance triggered by Toll-Interleukin1-Receptor domain (TIR)-family NLRs (TNLs). Transcriptional induction of the salicylic acid (SA) hormone defense sector provides one crucial barrier against biotrophic pathogens. Here, we present genetic and molecular evidence that in Arabidopsis an EDS1 complex with its partner PAD4 inhibits MYC2, a master regulator of SA-antagonizing jasmonic acid (JA) hormone pathways. In the TNL immune response, EDS1/PAD4 interference with MYC2 boosts the SA defense sector independently of EDS1-induced SA synthesis, thereby effectively blocking actions of a potent bacterial JA mimic, coronatine (COR). We show that antagonism of MYC2 occurs after COR has been sensed inside the nucleús but before or coincident with MYC2 binding to a target promoter, pANAC019. The stable interaction of PAD4 with MYC2 in planta is competed by EDS1-PAD4 complexes. However, suppression of MYC2-promoted genes requires EDS1 together with PAD4, pointing to an essential EDS1-PAD4 heterodimer activity in MYC2 inhibition. Taken together, these results uncover an immune receptor signaling circuit that intersects with hormone pathway crosstalk to reduce bacterial pathogen growth.

Reduced growth and viability is a common phenotype of plants with constitutively activated pathogen defenses. One branch of the plant innate immunity system, effector-triggered immunity, is especially potent and requires tight control to enable normal plant development. While some facets of this control that directly regulate resistance protein abundance or activity have been documented, general control of effector-triggered signaling sensitivity is poorly understood. We recently identified SUPPRESSOR OF rps4-RLD 1 (SRFR1), a novel negative regulator of avrRps4-triggered immunity. Mutations in SRFR1 were previously shown not to induce constitutive high expression of the defense gene PR1, and to be fully susceptible to the virulent Pseudomonas syringae pv. tomato strain DC3000. SRFR1 encodes a tetratricopeptide repeat-containing protein with weak similarity to transcriptional repressors in other organisms. By transient expression in Nicotiana benthamiana, SRFR1 was localized to the nucleus. Here we investigate more carefully whether expression of defense genes is misregulated in srfr1 mutant plants. Consistent with the hypothesized function of SRFR1 as a negative transcriptional regulator, we find that mRNA levels of several defense genes are upregulated in srfr1 mutants.