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SummaryBacterial pathogens are major causes of crop diseases, leading to significant production losses. For instance, kiwifruit canker, caused by the phytopathogen Pseudomonas syringae pv. actinidiae (Psa), has posed a global challenge to kiwifruit production. Treatment with copper and antibiotics, whilst initially effective, is leading to the rise of bacterial resistance, requiring new biocontrol approaches. Previously, we isolated a group of closely related Psa phages with biocontrol potential, which represent environmentally sustainable antimicrobials. However, their deployment as antimicrobials requires further insight into their properties and infection strategy. Here, we provide an in-depth examination of the genome of ΦPsa374-like phages and show that they use lipopolysaccharides (LPS) as their main receptor. Through proteomics and cryo-electron microscopy of ΦPsa374, we revealed the structural proteome and that this phage possess a T=9 capsid triangulation, unusual for myoviruses. Furthermore, we show that ΦPsa374 phage resistance arises in planta through mutations in a glycosyltransferase involved in LPS synthesis. Lastly, through in vitro evolution experiments we showed that phage-resistance is overcome by mutations in a tail fiber and structural protein of unknown function in ΦPsa374. This study provides new insight into the properties of ΦPsa374-like phages that informs their use as antimicrobials against Psa.Originality-Significance StatementThe rise of phytopathogen resistance to agrichemicals poses a significant threat to crop production, and requires urgent attention. The work presented here examines a phage genus, members of which utilize LPS as a receptor, and show potential as biocontrol agents of Psa. Studies in planta showed the development of Psa resistance to the phage, whilst maintaining bacterial virulence. This finding underscores the importance of examining phages in their ecological context for the informed design of phage cocktails that are effective in phytopathogen control.

SummaryTesting effector-knockout strains of thePseudomonas syringaepv.actinidiaebiovar 3 (Psa3) for reducedin plantagrowth in their native kiwifruit host revealed a number of non- redundant effectors that contribute to Psa3 pathogenicity. Conversely, complementation in the weak kiwifruit pathogenP. syringaepv.actinidifoliorum(Pfm) for increased growth identified redundant Psa3 effectors.Psa3 effectors hopAZ1a and HopS2b and the entire exchangeable effector locus (ΔEEL; 10 effectors) were significant contributors to bacterial colonisation of the host and were additive in their effects on pathogenicity. Four of the EEL effectors (HopD1a, AvrB2b, HopAW1a, and HopD2a) redundantly contribute to pathogenicity through suppression of pattern-triggered immunity (PTI).Important Psa3 effectors include several redundantly required effectors early in the infection process (HopZ5a, HopH1a, AvrPto1b, AvrRpm1a, and HopF1e). These largely target the plant immunity hub, RIN4.This comprehensive effector profiling revealed that Psa3 carries robust effector redundancy for a large portion of its effectors, covering a few functions critical to disease.

The complete genome of Pseudomonas syringae pv. actinidifoliorum ICMP18803 (Pfm) was sequenced using the Oxford Nanopore minION platform to an average read depth of 123. The genome assembled into a single chromosome of 6,353,853 bp after error-correction with Illumina short reads using Pilon. The complement of effector genes from a P. syringae pathovar plays the predominant role in defining its pathogenicity. Automatic gene annotation pipelines often poorly identify and name effector genes, however. Despite Pfm being a relatively weak pathogen of kiwifruit, a set of 31 effectors, 26 of which were full length, was identified by mapping the comprehensive effector library generated by Dillon et al. (2019). The Pfm genome with the effector complement, correctly named and annotated was resubmitted to Genbank (CP081457).

The infection of Pseudomonas syringae pv. actinidiae in kiwifruit is currently assessed by numerous methodologies, each with their own limitations. Most studies are based on either a laborious method of growth quantification of the pathogen or qualitative assessments by visual scoring following stem or cutting inoculation. Additionally, when assessing for resistance against specific pathogen effectors, confounding interactions between multiple genes in the pathogen can make mapping resistance phenotypes nearly impossible. Here, we present robust alternative methods to quantify pathogen load based on rapid bacterial DNA quantification by PCR, the use of Pseudomonas fluorescens, and a transient reporter eclipse assay for assessing resistance conferred by isolated bacterial avirulence genes. These assays compare well with bacterial plate counts to assess bacterial colonization as a result of plant resistance activation. The DNA-based quantification, when coupled with the P. fluorescens and reporter eclipse assays to independently identify bacterial avirulence genes, is rapid, highly reproducible, and scalable for high-throughput screens of multiple cultivars or genotypes. Application of these methodologies will allow rapid and high-throughput identification of resistant cultivars and the bacterial avirulence genes they recognize, facilitating resistance gene discovery for plant breeding programs.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Ralstonia solanacearum causes bacterial wilt disease in solanaceous crops. Identification of avirulence type III-secreted effectors recognized by specific disease resistance proteins in host plant species is an important step toward developing durable resistance in crops. In the present study, we show that R. solanacearum effector RipJ functions as an avirulence determinant in Solanum pimpinellifolium LA2093. In all, 10 candidate avirulence effectors were shortlisted based on the effector repertoire comparison between avirulent Pe_9 and virulent Pe_1 strains. Infection assays with transgenic strain Pe_1 individually carrying a candidate avirulence effector from Pe_9 revealed that only RipJ elicits strong bacterial wilt resistance in S. pimpinellifolium LA2093. Furthermore, we identified that several RipJ natural variants do not induce bacterial wilt resistance in S. pimpinellifolium LA2093. RipJ belongs to the YopJ family of acetyltransferases. Our sequence analysis indicated the presence of partially conserved putative catalytic residues. Interestingly, the conserved amino acid residues in the acetyltransferase catalytic triad are not required for effector-triggered immunity. In addition, we show that RipJ does not autoacetylate its lysine residues. Our study reports the identification of the first R. solanacearum avirulence protein that triggers bacterial wilt resistance in tomato. We expect that our discovery of RipJ as an avirulence protein will accelerate the development of bacterial wilt-resistant tomato varieties in the future. [Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY 4.0 International license .

Summary The common polysaccharide antigen (CPA) of the lipopolysaccharide (LPS) from Pseudomonas syringae is highly variable, but the genetic basis for this is poorly understood. We have characterized the CPA locus from P. syringae pv. actinidiae (Psa). This locus has genes for l‐ and d‐rhamnose biosynthesis and an operon coding for ABC transporter subunits, a bifunctional glycosyltransferase and an o‐methyltransferase. This operon is predicted to have a role in the transport, elongation and termination of the CPA oligosaccharide and is referred to as the TET operon. Two alleles of the TET operon were present in different biovars (BV) of Psa and lineages of the closely related pathovar P. syringae pv. actinidifoliorum. This allelic variation was reflected in the electrophoretic properties of purified LPS from the different isolates. Gene knockout of the TET operon allele from BV1 and replacement with that from BV3, demonstrated the link between the genetic locus and the biochemical properties of the LPS molecules in Psa. Sequence analysis of the TET operon from a range of P. syringae and P. viridiflava isolates displayed a phylogenetic history incongruent with core gene phylogeny but correlates with previously reported tailocin sensitivity, suggesting a functional relationship between LPS structure and tailocin susceptibility.

A pandemic isolate ofPseudomonas syringaepv.actinidiaebiovar 3 (Psa3) has devastated kiwifruit orchards growing cultivars ofActinidia chinensis. In contrast,A.arguta(kiwiberry) is not a host of Psa3. Resistance is mediated via effector-triggered immunity, as demonstrated by induction of the hypersensitive response in infectedA.argutaleaves, observed by microscopy and quantified by ion-leakage assays. Isolates of Psa3 that cause disease inA.argutahave been isolated and analyzed, revealing a 51 kb deletion in the exchangeable effector locus (EEL). This natural EEL-mutant isolate and strains with synthetic knockouts of the EEL were more virulent inA.argutaplantlets than wild-type Psa3. Screening of a complete library of Psa3 effector knockout strains identified increased growthin plantafor knockouts of four effectors–AvrRpm1a, HopF1c, HopZ5a, and the EEL effector HopAW1a –suggesting a resistance response inA.arguta. Hypersensitive response (HR) assays indicate that three of these effectors trigger a host species-specific HR. A Psa3 strain with all four effectors knocked out escaped host recognition, but a cumulative increase in bacterial pathogenicity and virulence was not observed. These avirulence effectors can be used in turn to identify the first cognate resistance genes inActinidiafor breeding durable resistance into future kiwifruit cultivars.

SummaryA pandemic isolate ofPseudomonas syringaepv.actinidiaebiovar 3 (Psa3) has devastated kiwifruit orchards growing cultivars ofActinidia chinensis. In contrast,A. arguta(kiwiberry) is resistant to Psa3. This resistance is mediated via effector-triggered immunity, as demonstrated by induction of the hypersensitive response in infectedA. argutaleaves, observed by microscopy and quantified by ion-leakage assays. Isolates of Psa3 that cause disease inA. argutahave been isolated and analyzed, revealing a 49 kb deletion in the exchangeable effector locus (EEL). This natural EEL-mutant isolate and strains with synthetic knockouts of the EEL were more virulent inA. argutaplantlets than wild-type Psa3. Screening of a complete library of Psa3 effector knockout strains identified increased growthin plantafor knockouts of four effectors – AvrRpm1a, HopF1c, HopZ5a, and the EEL effector HopAW1a – suggesting a resistance response inA. arguta. Hypersensitive response (HR) assays indicate that three of these effectors trigger a host species-specific HR. A Psa3 strain with all four effectors knocked out escaped host recognition, but a cumulative increase in bacterial pathogenicity and virulence was not observed. These avirulence effectors can be used in turn to identify the first cognate resistance genes inActinidiafor breeding durable resistance into future kiwifruit cultivars.

Bacterial wilt caused by the Ralstonia solanacearum species complex (RSSC) threatens the cultivation of important crops worldwide. We sequenced 30 RSSC phylotype I ( R. pseudosolanacearum ) strains isolated from pepper (Capsicum annuum) and tomato (Solanum lycopersicum) across the Republic of Korea. These isolates span the diversity of phylotype I, have extensive effector repertoires and are subject to frequent recombination. Recombination hotspots among South Korean phylotype I isolates include multiple predicted contact-dependent inhibition loci, suggesting that microbial competition plays a significant role in Ralstonia evolution. Rapid diversification of secreted effectors presents challenges for the development of disease-resistant plant varieties. We identified potential targets for disease resistance breeding by testing for allele-specific host recognition of T3Es present among South Korean phyloype I isolates. The integration of pathogen population genomics and molecular plant pathology contributes to the development of location-specific disease control and development of plant cultivars with durable resistance to relevant threats.

Bacterial wilt caused by theRalstonia solanacearumspecies complex (RSSC) threatens the cultivation of important crops worldwide. We sequenced 30 RSSC phylotype I (R. pseudosolanacearum) strains isolated from pepper (Capsicum annuum) and tomato (Solanum lycopersicum) across the Republic of Korea. These isolates span the diversity of phylotype I, have extensive effector repertoires and are subject to frequent recombination. Recombination hotspots among South Korean phylotype I isolates include multiple predicted contact-dependent inhibition loci, suggesting that microbial competition plays a significant role inRalstoniaevolution. Rapid diversification of secreted effectors presents challenges for the development of disease-resistant plant varieties. We identified potential targets for disease resistance breeding by testing for allele-specific host recognition of T3Es present among South Korean phyloype I isolates. The integration of pathogen population genomics and molecular plant pathology contributes to the development of location-specific disease control and development of plant cultivars with durable resistance to relevant threats.

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., The virulence contribution of CEL‐encoded effector AvrE1 and its phylogenetically related non‐CEL effector HopR1 from Psa3 are nonredundantly required for in planta Psa growth and lesion production on susceptible kiwifruit hosts.

The common polysaccharide antigen (CPA) from the lipopolysaccharide (LPS) component of cell walls from the species complex Pseudomonas syringae is highly variable both in structure and immunological specificity, but the genetic basis for this is not well understood. We have characterised the CPA locus from P. syringae pv. actinidiae (Psa). This locus has a modular structure with genes for both L- and D- rhamnose (Rha) biosynthesis and that of an unknown sugar. It also contains an operon coding for ABC transporter subunits, a bifunctional glycosyltransferase and an O-methyltransferase. This operon is predicted to have a role in transport, elongation and termination of the Rha backbone of the CPA oligosaccharide and is referred to as the TET operon. This is the first report of the identification of this operon in P. syringae. Two alleles of the TET operon were present amongst the different biovars of Psa and lineages of the closely related pathovar P. syringae pv. actinidifoliorum. This allelic variation was reflected in the electrophoretic properties of purified LPS from the different isolates. Gene knockout of the TET operon allele from biovar 1 and replacement with that from biovar 3, demonstrated the link between the genetic locus and the electrophoretic and immunogenic properties of the LPS molecules in Psa. Sequence analysis of the TET operon from a wide range of P. syringae and P. viridiflava isolates displayed a phylogenetic history which is incongruent with core gene phylogeny, but correlates with previously reported tailocin sensitivity, suggesting a functional relationship between LPS structure and tailocin susceptibility.

BackgroundPseudomonas syringaeis a widespread bacterial species complex that includes a number of significant plant pathogens. Amongst these,P. syringaepv.actinidiae(Psa) initiated a worldwide pandemic in 2008 on cultivars ofActinidia chinensisvar.chinensis. To gain information about the expression of genes involved in pathogenicity we have carried out transcriptome analysis ofPsaduring the early stages of kiwifruit infection.ResultsGene expression inPsawas 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.ConclusionsThe 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.

Plants evolved disease resistance (R) proteins that recognize corresponding pathogen effectors and activate effector-triggered immunity (ETI). However, it is largely unknown why, in some cases, a suppressor of ETI exists in plants. Arabidopsis SOBER1 (Suppressor of AvrBsT-elicited Resistance 1) was identified previously as a suppressor of Xanthomonas acetyltransferase effector AvrBsT-triggered immunity. Nevertheless, the extent to which SOBER1 suppresses ETI is unclear. Here, we identified SOBER1 as a suppressor of Pseudomonas acetyltransferase effector HopZ5-triggered immunity in Arabidopsis using recombinant inbred lines. Further analysis showed that SOBER1 suppresses immunity triggered by multiple bacterial acetyltransferases. Interestingly, SOBER1 interferes with the immunity signalling activated by some but not all tested acetyltransferase effectors, indicating that SOBER1 might target components that are shared between several ETI pathways.

Type-III secreted effectors (T3Es) play critical roles during bacterial pathogenesis in plants. Plant recognition of certain T3Es can trigger defence, often accompanied by macroscopic cell death, termed the hypersensitive response (HR). Economically important species of kiwifruit are susceptible to Pseudomonas syringae pv. actinidiae (Psa), the causal agent of kiwifruit bacterial canker. Although Psa is non-pathogenic in Arabidopsis thaliana, we observed that a T3E, HopZ5 that is unique to a global outbreak clade of Psa, triggers HR and defence in Arabidopsis accession Ct-1. Ws-2 and Col-0 accessions are unable to produce an HR in response to Pseudomonas-delivered HopZ5. While Ws-2 is susceptible to virulent bacterial strain Pseudomonas syringae pv. tomato DC3000 carrying HopZ5, Col-0 is resistant despite the lack of an HR. We show that HopZ5, like other members of the YopJ superfamily of acetyltransferases that it belongs to, autoacetylates lysine residues. Through comparisons to other family members, we identified an acetyltransferase catalytic activity and demonstrate its requirement for triggering defence in Arabidopsis and Nicotiana species. Collectively, data herein indicate that HopZ5 is a plasma membrane-localized acetyltransferase with autoacetylation activity required for avirulence.

The war between plants and their pathogens is endless, with plant resistance genes offering protection against pathogens until the pathogen evolves a way to overcome this resistance. Given how quickly new pathogen strains can arise and defeat plant defenses, it is critical to more rapidly identify and examine the specific genomic characteristics new virulent strains have gained which give them the upper hand. An indispensable tool is bioinformatics. Genome sequencing has advanced rapidly in the last decade, and labs are frequently uploading high-quality genomes of various organisms, including plant pathogenic bacteria such as Pseudomonas syringae. Pseudomonas syringae strains inject several effector proteins into host cells which often overcome host defenses. Probing online genomes provides a way to quickly and accurately predict effector repertoires of Pseudomonas, enabling the cloning of complete effector libraries of newly emerged strains. Here, we describe detailed protocols to rapidly clone bioinformatically predicted P. syringae effectors for various screening applications.

The war between plants and their pathogens is endless, with plant resistance genes offering protection against pathogens until the pathogen evolves a way to overcome this resistance. Given how quickly new pathogen strains can arise and defeat plant defenses, it is critical to more rapidly identify and examine the specific genomic characteristics new virulent strains have gained which give them the upper hand. An indispensable tool is bioinformatics. Genome sequencing has advanced rapidly in the last decade, and labs are frequently uploading high-quality genomes of various organisms, including plant pathogenic bacteria such as Pseudomonas syringae. Pseudomonas syringae strains inject several effector proteins into host cells which often overcome host defenses. Probing online genomes provides a way to quickly and accurately predict effector repertoires of Pseudomonas, enabling the cloning of complete effector libraries of newly emerged strains. Here, we describe detailed protocols to rapidly clone bioinformatically predicted P. syringae effectors for various screening applications.

Ralstonia solanacearum (Rso) is a causal agent of bacterial wilt disease in a wide range of horticultural crops. Rso strains are heterogeneous in nature and are therefore difficult in terms of both classification and development of disease resistance. Rso pathogen-associated molecular patterns (PAMPs) and effector proteins are secreted into plant cells, where they respectively activate and suppress plant immunity, thereby affecting Rso virulence. We review the current knowledge of Rso disease resistance and efforts to generate Rso-resistant crop plants. Further, we propose the introduction into plants of known pattern recognition receptors (PRRs) that recognize Rso PAMPs in order to confer resistance to a large number of strains. Additionally, the conserved ‘core’ effectors from Rso phylotypes could be used to identify and deploy nucleotide-binding leucine-rich repeat (NLR) resistance genes in a desired crop cultivar. We suggest that a phylotype-specific effector-assisted breeding program be instituted to rapidly identify disease resistance genes in available plant germplasm collections. Furthermore, stacking multiple NLRs that recognize Rso effectors would provide durable disease resistance by minimizing the chance for Rso to evade the implemented resistance. Finally, we propose that this strategy would most efficiently be achieved through development of transgenic crop lines.

The ability of plant tissues to retain totipotency despite being fully differentiated has been documented for decades. The transition from mature plant tissue to rejuvenated tissue first requires dedifferentiation of mature tissue, followed by rejuvenation (re-entry into the cell cycle) and somatic embryogenesis. We used a Nicotiana tabacum protoplast-based culture system to elucidate the role played by redox and phytohormone networks during the process of dedifferentiation and rejuvenation. Classical markers of redox homeostasis were measured during the rejuvenation process and lipid peroxidation is proposed as the best marker for indicating recovery of cells from oxidative stress sustained during the process of protoplast preparation and culture, prior to rejuvenation. A transient increase at 24 h after culture (HAC) in levels of a cytokinin riboside, iPA, suggests a putative novel function in initiating a stem-cell niche in an auxin dependent manner. A sharp rise at 72 HAC of gibberellin GA4, furthermore suggests a function for this hormone during the process of rejuvenation. These two key findings are consistent with previously described plant models for lateral root developmental. Therein, iPA could be involved in ‘stem-cell-niche’ initiation. Subsequently, GA4 could be involved in rapidly suppressing this initiation step following the earliest cell divisions, thereby enabling the establishment of this ‘niche’ into a callus-like tissue.