Summary Pathogen‐associated molecular patterns (PAMPs) are conserved molecules that are crucial for normal life cycle of microorganisms. However, the diversity of microbial PAMPs is little known. During screening of cell‐death‐inducing factors from the necrotrophic fungus Valsa mali, we identified a novel PAMP VmE02 that is widely spread in oomycetes and fungi. Agrobacterium tumefaciens‐mediated transient expression or infiltration of recombinant protein produced by Escherichia coli was performed to assay elicitor activity of the proteins tested. Virus‐induced gene silencing in Nicotiana benthamiana was used to determine the components involved in VmE02‐triggered cell death. The role of VmE02 in virulence and conidiation of V. mali were characterized by gene deletion and complementation. We found that VmE02, together with some of its homologues from both oomycete and fungal species, exhibited cell‐death‐inducing activity in N. benthamiana. VmE02‐triggered cell death was shown to be dependent on BRI1‐ASSOCIATED KINASE‐1, SUPPRESSOR OF BIR1‐1, HSP90 and SGT1 in N. benthamiana. Deletion of VmE02 in V. mali greatly attenuated pathogen conidiation but not virulence, and treatment of N. benthamiana with VmE02 enhances plant resistance to Sclerotinia sclerotiorum and Phytophthora capsici. We conclude that VmE02 is a novel cross‐kingdom PAMP produced by several fungi and oomycetes.

Plants use their innate immune system to defend against phytopathogens. As a part of this, pattern triggered-immunity is activated via pattern recognition receptor (PRR) detection of pathogen-associated molecular patterns (PAMPs). Although an increasing number of PAMPs have been identified, the PRRs for their recognition remain largely unknown. In the present study, we report a receptor-like protein RE02 (Response to VmE02) in Nicotiana benthamiana, which mediates the perception of VmE02, a PAMP previously identified from the phytopathogenic fungus Valsa mali, using virus-induced gene silencing (VIGS), co-immunoprecipitation, pull-down and microscale thermophoresis assays. We show that silencing of RE02 markedly attenuated VmE02-triggred cell death and immune responses. RE02 specifically interacted with VmE02 in vivo and in vitro, and it displayed a high affinity for VmE02. Formation of a complex with the receptor-like kinases SOBIR1 and BAK1 was essential for RE02 to perceive VmE02. Moreover, RE02-silenced plants exhibited enhanced susceptibility to both the oomycete Phytophthora capsici and the fungus Sclerotinia sclerotiorum, while overexpression of RE02 increased plant resistance to these pathogens. Together, our results indicate that the PAMP VmE02 and the receptor-like protein RE02 represent a new ligand-receptor pair in plant immunity, and that RE02 represents a promising target for engineering disease resistance.

Kiwifruit canker, caused by Pseudomonas syringae pv. actinidiae (Psa), is a destructive pathogen that globally threatens the kiwifruit industry. Understanding the molecular mechanism of plant-pathogen interaction can accelerate applying resistance breeding and controlling plant diseases. All known effectors secreted by pathogens play an important role in plant-pathogen interaction. However, the effectors in Psa and their function mechanism remain largely unclear. Here, we successfully identified a T3SS effector HopAU1 which had no virulence contribution to Psa, but could, however, induce cell death and activate a series of immune responses by agroinfiltration in Nicotiana benthamiana, including elevated transcripts of immune-related genes, accumulation of reactive oxygen species (ROS), and callose deposition. We found that HopAU1 interacted with a calcium sensing receptor in N. benthamiana (NbCaS) as well as its close homologue in kiwifruit (AcCaS). More importantly, silencing CaS by RNAi in N. benthamiana greatly attenuated HopAU1-triggered cell death, suggesting CaS is a crucial component for HopAU1 detection. Further researches showed that overexpression of NbCaS in N. benthamiana significantly enhanced plant resistance against Sclerotinia sclerotiorum and Phytophthora capsici, indicating that CaS serves as a promising resistance-related gene for disease resistance breeding. We concluded that HopAU1 is an immune elicitor that targets CaS to trigger plant immunity.

To successfully colonize the plants, the pathogenic microbes secrete a mass of effector proteins which manipulate host immunity. Apple valsa canker is a destructive disease caused by the weakly parasitic fungus Valsa mali. A previous study indicated that the V. mali effector protein 1 (VmEP1) is an essential virulence factor. However, the pathogenic mechanism of VmEP1 in V. mali remains poorly understood. In this study, we found that the apple (Malus domestica) pathogenesis-related 10 proteins (MdPR10) are the virulence target of VmEP1 using a yeast two-hybrid screening. By bimolecular fluorescence (BiFC) and coimmunoprecipitation (Co-IP), we confirmed that the VmEP1 interacts with MdPR10 in vivo. Silencing of MdPR10 notably enhanced the V. mali infection, and overexpression of MdPR10 markedly reduced its infection, which corroborates its positive role in plant immunity against V. mali. Furthermore, we showed that the co-expression of VmEP1 with MdPR10 compromised the MdPR10-mediated resistance to V. mali. Taken together, our results revealed a mechanism by which a V. mali effector protein suppresses the host immune responses by interfering with the MdPR10-mediated resistance to V. mali during the infection.

Necrosis and ethylene-inducing peptide 1(Nep1)-like protein (NLP) is well known for its cytotoxicity and immunogenicity on dicotyledonous, and it has attracted large attention due to its gene expansion and functional diversification in numerous phytopathogens. Here, two NLP family proteins, VmNLP1 and VmNLP2, were identified in the pathogenic fungus Valsa mali. We showed that VmNLP2 but not VmNLP1 induced cell death when transiently expressed in Nicotiana benthamiana. VmNLP2 was also shown to induce cell death in apple leaves via the treatment of the Escherichia coli-produced recombinant protein. VmNLP1 and VmNLP2 transcripts were drastically induced at the early stage of V. mali infection, whereas only VmNLP2 was shown to be essential for pathogen virulence. We also found that VmNLP1 and VmNLP2 are required for maintaining the integrity of cell membranes, and they differentially contribute to V. mali tolerance to salt- and osmo-stresses. Notably, multiple sequence alignment revealed that the second histidine (H) among the conserved heptapeptide (GHRHDWE) of VmNLP2 is mutated to tyrosine (Y). When this tyrosine (Y) was substituted by histidine (H), the variant displayed enhanced cytotoxicity in N. benthamiana, as well as enhanced virulence on apple leaves, suggesting that the virulence role of VmNLP2 probably correlates to its cytotoxicity activity. We further showed that the peptide among VmNLP2, called nlp25 (VmNLP2), triggered strong immune response in Arabidopsis thaliana. This work demonstrates that NLPs from V. mali involve multiple biological roles, and shed new light on how intricately complex the functions of NLP might be.