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AvrE1 and HopR1 from Pseudomonas syringae pv. actinidiae are additively required for full virulence on kiwifruit.

ABSTRACT: Pseudomonas syringae pv. actinidiae ICMP 18884 biovar 3 (Psa3) produces necrotic lesions during infection of its kiwifruit host. Bacterial growth in planta and lesion formation are dependent upon a functional type III secretion system (T3S), which translocates multiple effector proteins into host cells. Associated with the T3S locus is the conserved effector locus (CEL), which has been characterized and shown to be essential for the full virulence in other P. syringae pathovars. Two effectors at the CEL, hopM1 and avrE1, as well as an avrE1-related non-CEL effector, hopR1, have been shown to be redundant in the model pathogen P. syringae pv. tomato DC3000 (Pto), a close relative of Psa. However, it is not known whether CEL-related effectors are required for Psa pathogenicity. The Psa3 allele of hopM1, and its associated chaperone, shcM, have diverged significantly from their orthologs in Pto. Furthermore, the CEL effector hopAA1-1, as well as a related non-CEL effector, hopAA1-2, have both been pseudogenized. We have shown that HopM1 does not contribute to Psa3 virulence due to a truncation in shcM, a truncation conserved in the Psa lineage, probably due to the need to evade HopM1-triggered immunity in kiwifruit. We characterized the virulence contribution of CEL and related effectors in Psa3 and found that only avrE1 and hopR1, additively, are required for in planta growth and lesion production. This is unlike the redundancy described for these effectors in Pto and indicates that these two Psa3 genes are key determinants essential for kiwifruit bacterial canker disease.

SUBMITTER: Jayaraman J

PROVIDER: S-EPMC7548996 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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AvrE1 and HopR1 from Pseudomonas syringae pv. actinidiae are additively required for full virulence on kiwifruit.

Jayaraman Jay J Yoon Minsoo M Applegate Emma R ER Stroud Erin A EA Templeton Matthew D MD

Molecular plant pathology 20200923 11

Pseudomonas syringae pv. actinidiae ICMP 18884 biovar 3 (Psa3) produces necrotic lesions during infection of its kiwifruit host. Bacterial growth in planta and lesion formation are dependent upon a functional type III secretion system (T3S), which translocates multiple effector proteins into host cells. Associated with the T3S locus is the conserved effector locus (CEL), which has been characterized and shown to be essential for the full virulence in other P. syringae pathovars. Two effectors at ...[more]

PMID: 32969167

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Project description:BACKGROUND:Pseudomonas syringae is a widespread bacterial species complex that includes a number of significant plant pathogens. Amongst these, P. syringae pv. actinidiae (Psa) initiated a worldwide pandemic in 2008 on cultivars of Actinidia chinensis var. chinensis. To gain information about the expression of genes involved in pathogenicity we have carried out transcriptome analysis of Psa during the early stages of kiwifruit infection. RESULTS:Gene expression in Psa was investigated during the first five days after infection of kiwifruit plantlets, using RNA-seq. Principal component and heatmap analyses showed distinct phases of gene expression during the time course of infection. The first phase was an immediate transient peak of induction around three hours post inoculation (HPI) that included genes that code for a Type VI Secretion System and nutrient acquisition (particularly phosphate). This was followed by a significant commitment, between 3 and 24 HPI, to the induction of genes encoding the Type III Secretion System (T3SS) and Type III Secreted Effectors (T3SE). Expression of these genes collectively accounted for 6.3% of the bacterial transcriptome at this stage. There was considerable variation in the expression levels of individual T3SEs but all followed the same temporal expression pattern, with the exception of hopAS1, which peaked later in expression at 48 HPI. As infection progressed over the time course of five days, there was an increase in the expression of genes with roles in sugar, amino acid and sulfur transport and the production of alginate and colanic acid. These are both polymers that are major constituents of extracellular polysaccharide substances (EPS) and are involved in biofilm production. Reverse transcription-quantitative PCR (RT-qPCR) on an independent infection time course experiment showed that the expression profile of selected bacterial genes at each infection phase correlated well with the RNA-seq data. CONCLUSIONS:The results from this study indicate that there is a complex remodeling of the transcriptome during the early stages of infection, with at least three distinct phases of coordinated gene expression. These include genes induced during the immediate contact with the host, those involved in the initiation of infection, and finally those responsible for nutrient acquisition.

Project description:Pseudomonas syringae pv. actinidiae (Psa), the causal agent of kiwifruit canker, is one of the most devastating plant diseases of recent times. We have generated two mini-Tn5-based random insertion libraries of Psa ICMP 18884. The first, a 'phenotype of interest' (POI) library, consists of 10,368 independent mutants gridded into 96-well plates. By replica plating onto selective media, the POI library was successfully screened for auxotrophic and motility mutants. Lipopolysaccharide (LPS) biosynthesis mutants with 'Fuzzy-Spreader'-like morphologies were also identified through a visual screen. The second, a 'mutant of interest' (MOI) library, comprises around 96,000 independent mutants, also stored in 96-well plates, with approximately 200 individuals per well. The MOI library was sequenced on the Illumina MiSeq platform using Transposon-Directed Insertion site Sequencing (TraDIS) to map insertion sites onto the Psa genome. A grid-based PCR method was developed to recover individual mutants, and using this strategy, the MOI library was successfully screened for a putative LPS mutant not identified in the visual screen. The Psa chromosome and plasmid had 24,031 and 1,236 independent insertion events respectively, giving insertion frequencies of 3.65 and 16.6 per kb respectively. These data suggest that the MOI library is near saturation, with the theoretical probability of finding an insert in any one chromosomal gene estimated to be 97.5%. However, only 47% of chromosomal genes had insertions. This surprisingly low rate cannot be solely explained by the lack of insertions in essential genes, which would be expected to be around 5%. Strikingly, many accessory genes, including most of those encoding type III effectors, lacked insertions. In contrast, 94% of genes on the Psa plasmid had insertions, including for example, the type III effector HopAU1. These results suggest that some chromosomal sites are rendered inaccessible to transposon insertion, either by DNA-binding proteins or by the architecture of the nucleoid.

Project description: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.

Dataset Information

AvrE1 and HopR1 from Pseudomonas syringae pv. actinidiae are additively required for full virulence on kiwifruit.

Publications

AvrE1 and HopR1 from Pseudomonas syringae pv. actinidiae are additively required for full virulence on kiwifruit.

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