Two NIS1-like proteins from apple canker pathogen (Valsa mali) play distinct roles in plant recognition and pathogen virulence.

Conserved effectors produced by phytopathogens play critical roles in plant-microbe interactions. NIS1-like proteins represent a newly identified family of effectors distributed in multiple fungal species. However, their biological functions in a majority of pathogenic fungi remain largely elusive and require further investigation. In this study, we characterized two NIS1-like proteins VmNIS1 and VmNIS2 from Valsa mali, the causal agent of apple Valsa canker. Both of these two proteins were predicted to be secreted. Using agroinfiltration, we found that VmNIS1 induced intense cell death, whereas VmNIS2 suppressed INF1 elicitin-triggered cell death in Nicotiana benthamiana. Treatment of N. benthamiana with VmNIS1 recombinant protein produced by Escherichia coli activated a series of immune responses and enhanced plant disease resistance against Phytophthora capsici. In contrast, VmNIS2 suppressed plant immune responses and promoted P. capsici infection when transiently expressed in N. benthamiana. Both VmNIS1 and VmNIS2 were shown to be highly induced at late stage of V. mali infection. By individually knocking out of these two genes in V. mali, however, only VmNIS2 was shown to be required for pathogen virulence as well as tolerance to oxidative stress. Notably, we further showed that C-terminal extension of VmNIS1 was essential for plant recognition and VmNIS2 may escape plant detection via sequence truncation. Our data collectively indicate that VmNIS1 and VmNIS2 play distinct roles in plant recognition and pathogen virulence, which provided new insights into the function of NIS1-like proteins in plant-microbe interactions.