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.

MicroRNA-like RNAs (milRNAs) are a class of regulatory factors commonly found in fungi with similar generation and action mechanism as plant and animal microRNAs, which are widely involved in their growth and development, as well as in life activities such as infection and pathogenesis of plant pathogenic fungi. The apple Valsa canker caused by Valsa mali is the most destructive disease affecting apple production.

The research aims to explore the function and mechanism of Vm-milRN7 in regulating the pathogenicity of V. mali, and to provide a theoretical basis for targeted disease resistance breeding of apple Valsa canker.

Vm-milRN7 precursor overexpression vector was constructed by amplifying Vm-milRN7 precursor sequence using genomic DNA of strain 03-8 as a template; upstream and downstream sequences of Vm-milRN7 precursor were amplified and Vm-milRN7 precursor knockout mutants were constructed by using Double-joint PCR technique. The Vm-milRN7 precursor overexpression strains and knockout mutants were constructed by PEG-mediated protoplast transformation. The vegetative growth rate of Vm-milRN7 precursor overexpression strains and knockout mutants was determined by cultivation on PDA medium, and the pathogenicity of these strains was verified by inoculation on apple twigs and leaves. The regulatory relationship between Vm-milRN7 and its potential target gene Vm-09496 was identified by qRT-PCR and co-infiltration experiment in Nicotiana benthamiana leaves; protein sequence characterization and phylogenetic analysis of Vm-09496 were performed using bioinformatics software. In order to analyze its function, Vm-09496 knockout mutants and complement strains were created and their phenotypes were characterized.

PEG-mediated genetic transformation was used to create Vm-milRN7 overexpression strains and knockout mutants. Vm-milRN7 overexpression strains showed no apparent alteration in vegetative growth rate compared with that of the wild-type strains, whereas the knockout mutants had a significantly lower vegetative growth rate. Compared with the wild-type strains, the overexpression strains showed a significant increase in the pathogenicity of V. mali on apple leaves, while the knockout mutants showed a significant decrease in the pathogenicity of V. mali on apple leaves and twigs. Further, qRT-PCR and co-infiltration in N. benthamiana leaves assay showed that Vm-milRN7 inhibited the expression of its potential target gene Vm-09496. Bioinformatics analysis revealed that the gene encodes an imaginary protein and is evolutionarily most closely related to VP1G_09956 in V. pyri. In order to analyze its function, the Vm-09496 knockout mutants and complement strains were created, and the pathogenicity of the knockout mutants on both twigs and leaves was significantly increased compared with that of the wild-type strain, while the vegetative growth rate and pathogenicity of the complement strains recovered to the wild-type level.

Vm-milRN7 may be involved in the pathogenity of V. mali by regulating the expression of the target gene Vm-09496. The target gene Vm-09496 is an important endogenous gene affecting V. mali infection and negatively regulates V. mali pathogenicity.