Scientia Agricultura Sinica ›› 2022, Vol. 55 ›› Issue (3): 503-513.doi: 10.3864/j.issn.0578-1752.2022.03.007

• PLANT PROTECTION • Previous Articles     Next Articles

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

ZHANG JinLong(),ZHAO ZhiBo,LIU Wei,HUANG LiLi()   

  1. College of Plant Protection, Northwest A&F University/State Key Laboratory of Crop Stress Biology for Arid Areas, Yangling 712100, Shaanxi
  • Received:2021-07-14 Accepted:2021-08-26 Online:2022-02-01 Published:2022-02-11
  • Contact: LiLi HUANG E-mail:535268139@qq.com;huanglili@nwsuaf.edu.cn

Abstract: 【Objective】 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.【Method】 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.【Result】 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.【Conclusion】 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.

Key words: bacterial canker of kiwifruit, Pseudomonas syringae pv. actinidiae (Psa), effector, T3SS, pathogenicity

Table 1

The list of the detail information of deletion and complement mutants"

突变体名称
The name of mutants
详细信息
Related detail information
ΔhrcC T3SS结构基因hrcC功能缺陷突变体 hrcC-deletion generating the T3SS-deficient mutant
ΔhrcS T3SS结构基因hrcS功能缺陷突变体hrcS-deletion generating the T3SS-deficient mutant
ΔO T3SE hopM1/avrE1缺失突变体T3SE hopM1/avrE1-deletion mutant
ΔM T3SE hopR1缺失突变体T3SE hopR1-deletion mutant
ΔOM T3SE hopM1/avrE1+hopR1缺失突变体T3SE hopR1-deletion mutant based on ΔO background
ΔMO T3SE hopR1+hopM1/avrE1缺失突变体T3SE hopM1/avrE1-deletion mutant based on ΔM background
ΔM-C-hopR1 T3SE hopR1突变体基础上回补hopR1的回补菌株T3SE hopR1-complement mutant based on ΔM background
ΔOM-C-hopR1 T3SE hopR1+hopM1/AvrE1突变体基础上回补hopR1的回补菌株T3SE hopR1-complement mutant based on ΔOM background
ΔA 表2中对应T3SE基因簇A缺失突变体T3SE cluster A-deletion mutant listing on Table 2
ΔF 表2中对应T3SE基因簇F缺失突变体T3SE cluster F-deletion mutant listing on Table 2
ΔE 表2中对应T3SE基因簇E缺失突变体T3SE cluster E-deletion mutant listing on Table 2
ΔAF 表2中对应T3SE基因簇A和F缺失突变体T3SE cluster A- and F-deletion mutant listing on Table 2
ΔAFE 表2中对应T3SE基因簇A、F和E全敲除缺失突变体T3SE cluster A-, F- and E-deletion mutant listing on Table 2
ΔAFEL 在ΔAFE突变体的背景下敲除avrPto5获得突变体avrPto5-deletion mutant based on ΔAFE background
ΔAFELM 在ΔAFEL突变体的背景下敲除hopR1获得突变体hopR1-deletion mutant based on ΔAFEL background
ΔAFELO 在ΔAFEL突变体的背景下敲除hopM1/avrE1获得突变体hopM1/avrE1-deletion mutant based on ΔAFEL background
ΔAFELOM 在ΔAFEL突变体的背景下同时敲除hopM1/avrE1hopR1获得突变体
hopM1/avrE1- and hopR1-deletion mutant based on ΔAFEL background
ΔOMN 在ΔOM突变体基础上敲除avrRpm1获得突变体avrRpm1-deletion mutant based on ΔOM background

Table 2

The T3SE repertoire of M228 and M227 strains of Psa"

位置
Locus
T3SE ICMP 18884 伴侣分子
Chaperon
M228 M227 基因敲除标识符
Symbol of gene deletion
HopY1 IYO_000845
Cluster A HopQ1 IYO_003525 A
HopD1 IYO_003530
AvrD1 IYO_003570
AvrB4-l IYO_003600
HopAYl IYO_003610
HopX3 IYO_003635 ShcF
HopAW1 IYO_003657
HopBB1-2 IYO_003675 ShcF
HopAF1 IYO_003680
HopAO2 IYO_003720
HopBB1-1 IYO_003727 ShcF
HopS2 IYO_004052 ShcS2
HopI1 IYO_005160
CEL/Cluster O HopN1 IYO_006735 ShcN
HopAA1-1 IYO_006745
HopM1 IYO_006760 ShcM O
AvrE1 IYO_006770 ShcE
AvrRpm1 IYO_008065 N
Cluster E HopZ5 IYO_008282 E
HopH1 IYO_008285
HopAM1-2 IYO_008385
HopAE1 IYO_012225
HopAZ1 IYO_018555
AvrPto5 IYO_020425 L
HonAM1-1 IYO_023205
Cluster F HopAI1 IYO_023980 F
HopAH1 IYO_023985
HopAG1 IYO_023990
Cluster M HopR1 IYO_024150 M
HopW1 IYO_024160
HopF2 IYO_024217 ShcF
HopAS1 IYO_027420
HopA1 IYO_028375 ShcA
HopZ3 IYO_029045 Tir
AvrRpm2 IYO_029288 ShcF
Plasmid HopAV1 IYO_029710
HopAA1-2 IYO_029780-795
HopAU1 IYO_029795
存在的基因 Present 差异的基因 Variable 不完整的基因 Incomplete

Fig. 1

The T3SS-deficient mutants of Psa can’t induce symptoms on kiwifruit, and elicit the HR in N. benthamina leaves"

Fig. 2

The ∆hopM1/avrE1 mutants showed significantly reduced pathogenicity 试验至少重复2次,得相似结果Experiments were repeated at least twice with similar results (Student’s t-test,P<0.01)The lesion length for each strain was normalized to that of the control strain M228. The same as below"

Fig. 3

The hopR1-deletion mutants showed significantly reduced pathogenicity"

Fig. 4

The redundant effector group is present in three T3SEs cluster A, E and F within M228 The pathogenicity of different strains was compared after inoculated on the same variety (Duncan’s multiple range test, P<0.05). The same as Fig. 5"

Fig. 5

The AvrPto5- and AvrRpm1-deletion mutants showed significantly increased pathogenicity on kiwifruitThe length of lesion expansion of the above mutants was measured 15 days post inoculation"

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