Genetic Causes of Non-pathogenic Pseudomonas syringae pv. actinidiae Isolates in Kiwifruit Orchards

Abstract

Bacterial canker disease has become the largest threat to kiwifruit cultivation and production. A monomorphic subpopulation of Pseudomonas syringae pv. actinidiae biovar 3 (Psa3) is responsible for the pandemic worldwide. Diversity in pathogenicity has been found in the pandemic subpopulation and in other Psa3 subpopulations causing epidemics in China. However, the genetic bases have not yet been elucidated. In this study, 117 Psa3 isolates were identified by Psa- and Psa3-specific primers, and evaluated for pathogenicity. Three isolates G4, G40, and S2 are not pathogenic to kiwifruit and do not elicit hypersensitivity responses (HRs) in non-host Nicotiana benthamiana leaves. Two isolates, G25 and G35, exhibited attenuated HR-eliciting activity in non-host N. benthamiana, but they exhibited greatly and slightly reduced pathogenicity in host plants, respectively. The genomes of the five isolates were sequenced and compared with closely related isolates revealed by MLVA and whole-genome typing methods. The candidate genetic loci responsible for the changes in pathogenicity and HR elicitation, were further evaluated by allele replacement experiments. We found that the three non-pathogenic isolates were formed due to the independent, identical insertion events of ISPsy36 transposon in the hrpR gene, encoding a key regulator of type III secretion system (T3SS) and type III effectors (T3Es). In the symptomatic sample from which G4 was isolated, 27% HR negative isolates were detected. In isolate G25, transposon insertion of ISPsy32 at the non-coding sequence upstream of the hrpR gene was detected, similar to a previously reported low-virulent Psa3 strain M227. In isolate G35, we detected disruptions of T3Es hopBB1-1 and hopBB1-2, which induce HR in N. benthamiana leaves revealed by Agrobacterium tumefaciens infiltration. These phenotype-changed isolates were formed at low frequencies during the course of pathogen infection in host plants, supported by the binding assay of ISPsy32 and the non-coding DNA sequences upstream of the hrpR gene, the co-isolation of the virulent isolates belonging to the same MLVA clade, and the low levels of transcription of the transposon genes. Taken together, in terms of short-term field evolution, transposon insertions in the T3SS-related genes resulted in the formation of non-pathogenic and low-virulent Psa3 isolates.

The OmpR-like Transcription Factor as a Negative Regulator of hrpR/S in Pseudomonas syringae pv. actinidiae

“…Transcriptome and qRT-PCR results showed that OmpR-like deletion led to an increase in hrpR/S transcription, while its overexpression led to a decrease in hrpR/S transcription, suggesting that OmpR-like can directly and negatively regulate hrpR/S transcription and the expression of downstream T3SS genes. Our DNA pull-down data (ProteomeXchange accession: PXD023722) [ 33 ] further supported the direct interaction between the OmpR-like protein and the promoter region of the hrpR/S gene. A recent study by Shao et al [ 40 ] also demonstrated that EnvZ-OmpR is a repressor of the T3SS in P. savastanoi pv.…”

“…Previously, we performed a DNA pull-down assay using the biotin-labeled DNA sequence upstream of hrpR/S gene as a probe to detect the protein candidates potentially regulating the transcription of hrpR/S gene and found an OmpR-like transcription factor (CN228_13375, GenBank accession: AYL80818.1) as a highly reliable candidate (ProteomeXchange accession: PXD023722) [ 33 ]. In this study, the regulatory mechanism and other functions of the OmpR-like transcription factor, involved in regulating the hrpR/S gene in Psa , were explored by techniques such as gene knockout and overexpression, ChIP-seq, and RNA-seq.…”

Genomic Variation and Host Interaction among Pseudomonas syringae pv. actinidiae Strains in Actinidia chinensis ‘Hongyang’

“…A similar case was found in the M227 strain isolated in China: a transposable insertion at the 930 bp upstream of HrpR caused reduced pathogenicity by attenuating the expression of TTSS and TTEs [ 17 ]. For the other three non-pathogenic strains (S2, G4, G40), the ISPsy36 transposon insertion was found at the 573 bp position of HrpR [ 30 ]. In the present work, the HrpR gene of two low-pathogenicity strains, ScPzH and ScDjyH, was interrupted at 596 bp, resulting in a mutated gene encoding a truncated protein ( Figure 3 A).…”

Transcriptomic and Proteomic Profiling Reveal the Key Role of AcMYB16 in the Response of Pseudomonas syringae pv. actinidiae in Kiwifruit

“…Kiwifruit bacterial canker (KBC), caused by Pseudomonas syringae pv. actinidiae ( Psa ), has already become an economic threat to the kiwifruit industry worldwide ( Tahir et al, 2019 ; Li et al, 2021 ). Many efforts have been made to uncover the population structure of Psa , and at least five types of populations, including biovars 1, 2, 3, 5, and 6, have been defined based on their virulence, toxin production, and host range, of which biovar 3 is responsible for the global pandemic ( He et al, 2019 ).…”