The Function of Key T3SS Effectors in Pseudomonas syringae pv. actinidiae

Pseudomonas syringae pv. actinidiae (Psa), the causal agent of bacterial canker of kiwifruit, is the most devastating pathogen in global kiwifruit production. The pathogenic bacteria secrete a series of effectors (T3SEs) into host cell to promote infection and pathogenesis by the type III secretion system (T3SS). The objective of this study is to analyze the T3SEs information in Psa genome and systematically evaluate the pathogenicity function of T3SS and T3SEs, and to provide the basis evidence for the research of the pathogenic mechanism and the establishment of the control strategies.

By marker-free homologous recombination knockout technique, the M228 deficiency mutants of T3SS, ΔhrcS and ΔhrcC, were obtained for inoculating on host to evaluate the pathogenicity and injecting on Nicotiana benthamiana to observe the cell death response. Then, based on the T3SEs database downloaded from Pseudomonas-Plant Interaction, the T3SEs library of strong pathogenicity M228 and weak pathogenicity M227 was separately constructed against the database by local BLAST multiple sequence alignment program, and then the T3SEs information between them was compared. Moreover, 20 T3SE single- and poly-genetic mutants from M228 and 2 HopR1-genetic complementing mutants were constructed, involving 19 T3SEs, and then the mutants were wound-inoculated on kiwifruit canes for assessing and statistical analyzing the pathogenicity.

T3SS was proved to be essential for Psa pathogenicity on host and hypersensitive response (HR) on non-host by the hrcS and hrcC mutants, separately. Further the BLAST results against database showed there were almost 31 complete T3SE genes and their sequences were displayed 100% similarity between the strong pathogenicity strain and attenuated strain. Then, some T3SE genes were selected for deletion mutants. The results showed that hopM1/avrE1 and hopR1 genes were essential for Psa pathogenicity and had no function redundant with each other. In addition, the avrPto5- and avrRpm1-deletion mutant could in turn increase the Psa pathogenicity. Based on avrPto- and T3SE group (cluster A, E and F) deletion mutant, single- or poly-genetic mutant of hopR1 and hopM1/avrE1 could still separately lead to a significant decrease in Psa pathogenicity. However, simultaneous deletion of hopM1/avrE1, hopR1, avrPto5 and A-F-E cluster resulted in complete loss of pathogenicity.

HopR1 and its homologous family HopM1/AvrE1, which don't have a redundant function independent with others, are the unique key pathogenicity factors in Psa, but AvrPto5- and AvrRpm1-deletion can enhance Psa pathogenicity.