Biology subjects

Jayaraman, J.

Publications and source records attributed to Jayaraman, J..

11 recordsLinked to original sources

bioRxiv2025

Diverse Pseudomonas species engage in beneficial and suppressive interactions with the kiwifruit pathogen Psa across Actinidia germplasm

In 2010, a Pseudomonas syringae pv. actinidiae biovar 3 (Psa3) incursion into New Zealand kiwifruit orchards devastated susceptible Actinidia chinensis cultivars. In contrast, many Actinidia species maintained in germplasm collections were resistant to Psa3 and showed limited symptoms. Recent genome biosurveillance revealed the emergence of widespread leaf spot symptoms in Psa3-resistant Actinidia germplasm. Surprisingly, few Psa3 isolates were recovered from symptomatic tissues, despite the frequent isolation of phenotypical Pseudomonas isolates on selective agar. Despite the poor recovery of Psa3 isolates, Psa3 was found in all symptomatic leaf tissue through qPCR and metabarcoding analysis. Metabarcoding revealed stark differences in bacterial community composition from samples taken from lesion-carrying or lesion-free material from the same leaf. Host genotype also appeared to influence community composition but to a lesser extent. Whole genome sequencing of diverse Pseudomonas spp. isolates revealed that many belonged to the P. syringae species complex. Curiously, the kiwifruit-associated P. syringae pv. actinidifoliorum (Pfm), was never recovered, nor were any other phylogroup 1 pathovars. Pathogenicity and competitive assays revealed that while individually diverse Pseudomonas isolates were not more pathogenic than Pfm or Psa3 on resistant Actinidia hosts, they could interact with Psa to sometimes improve their growth, suggesting that these isolates may form pathogenic consortia in these disease-associated phyllosphere communities on typically Psa3-resistant hosts. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=116 SRC="FIGDIR/small/635981v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@1e48d68org.highwire.dtl.DTLVardef@1ffa8d7org.highwire.dtl.DTLVardef@1a5c47corg.highwire.dtl.DTLVardef@6bd39_HPS_FORMAT_FIGEXP M_FIG C_FIG

plant biology↗

bioRxiv2024

Genomic biosurveillance of the kiwifruit pathogen Pseudomonas syringae pv. actinidiae biovar 3 reveals adaptation to selective pressures in New Zealand orchards

In the late 2000s, a pandemic of Pseudomonas syringae pv. actinidiae biovar 3 (Psa3) devastated kiwifruit orchards growing susceptible yellow-fleshed cultivars. New Zealands kiwifruit industry has since recovered, following the deployment of the tolerant cultivar Zesy002. However, little is known about the extent to which the Psa population is evolving since its arrival. Over 500 Psa3 isolates from New Zealand kiwifruit orchards were sequenced between 2010 and 2022, from commercial monocultures and diverse germplasm collections. While effector loss was previously observed on Psa-resistant germplasm vines, effector loss appears to be rare in commercial orchards, where the dominant cultivars lack Psa resistance. However, a new Psa3 variant, which has lost the effector hopF1c, has arisen. The loss of hopF1c appears to have been mediated by the movement of integrative conjugative elements introducing copper resistance into this population. Following this variants identification, in planta pathogenicity and competitive fitness assays were performed to better understand the risk and likelihood of its spread. While hopF1c loss variants had similar in planta growth to wild-type Psa3, a lab-generated {Delta}hopF1c strain could outcompete wild-type on select hosts. Further surveillance was conducted in commercial orchards where these variants were originally isolated, with 6.6% of surveyed isolates identified as hopF1c loss variants. These findings suggest that the spread of these variants is currently limited, and they are unlikely to cause more severe symptoms than the current population. Ongoing genome biosurveillance of New Zealands Psa3 population is recommended to enable early detection and management of variants of interest.

genomics↗

bioRxiv2024

Identification and characterization of innate immunity in Actinidia melanandra in response to Pseudomonas syringae pv. actinidiae

Pseudomonas syringae pv. actinidiae biovar 3 (Psa3) has decimated kiwifruit orchards growing susceptible kiwifruit Actinidia chinensis varieties. Effector loss has occurred recently in Psa3 isolates from resistant kiwifruit germplasm, resulting in strains capable of partially overcoming resistance present in kiwiberry vines (A. arguta, A. polygama, and A. melanandra). Diploid male A. melanandra recognises several effectors, sharing recognition of at least one avirulence effector (HopAW1a) with previously studied tetraploid kiwiberry vines. Sequencing and assembly of the A. melanandra genome enabled the characterisation of the transcriptomic response of this non-host to wild-type and genetic mutants of Psa3. A. melanandra appears to mount a classic effector-triggered immunity (ETI) response to wildtype Psa3 V-13, as expected. Surprisingly, the type III secretion (T3S) system-lacking Psa3 V-13 {Delta}hrcC strain did not appear to trigger pattern-triggered immunity (PTI) despite lacking the ability to deliver immunity-suppressing effectors. Contrasting the A. melanandra responses to an effectorless Psa3 V-13 {Delta}33E strain and to Psa3 V-13 {Delta}hrcC suggested that PTI triggered by Psa3 V-13 was based on the recognition of the T3S itself. The characterisation of both ETI and PTI branches of innate immunity responses within A. melanandra further enables breeding for durable resistance in future kiwifruit cultivars.

plant biology↗

bioRxiv2023

NbPTR1 confers resistance against Pseudomonas syringae pv. actinidiae in kiwifruit

Pseudomonas syringae pv. actinidiae biovar 3 (Psa3) causes a devastating canker disease in yellow-fleshed kiwifruit (Actinidia chinensis). The effector HopZ5, which is present in all isolates of Psa3 causing global outbreaks of pandemic kiwifruit canker disease, triggers immunity in Nicotiana benthamiana and is not recognised in susceptible A. chinensis cultivars. In a search for N. benthamiana non-host resistance genes against HopZ5, we found that the nucleotide-binding leucine-rich repeat receptor NbPTR1 recognised HopZ5. RPM1-interacting protein 4 (RIN4) orthologues from multiple plants, including kiwifruit, were associated with NbPTR1-mediated autoimmunity suppression and recognition of HopZ5. No functional orthologues of NbPTR1 were found in A. chinensis. NbPTR1 transformed into Psa3-susceptible A. chinensis var. chinensis Hort16A plants introduced HopZ5-specific resistance against Psa3. Altogether, this study suggested that expressing NbPTR1 in Psa3-susceptible kiwifruit is a viable approach to acquiring resistance to Psa3 and it provides valuable information for engineering resistance in otherwise susceptible kiwifruit genotypes.

plant biology↗

bioRxiv2022

Contrasting effector profiles between bacterial colonisers of kiwifruit reveal redundant roles and interplay converging on PTI-suppression and RIN4

O_LITesting effector-knockout strains of the Pseudomonas syringae pv. actinidiae biovar 3 (Psa3) for reduced in planta growth in their native kiwifruit host revealed a number of non- redundant effectors that contribute to Psa3 pathogenicity. Conversely, complementation in the weak kiwifruit pathogen P. syringae pv. actinidifoliorum (Pfm) for increased growth identified redundant Psa3 effectors. C_LIO_LIPsa3 effectors hopAZ1a and HopS2b and the entire exchangeable effector locus ({Delta}EEL; 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). C_LIO_LIImportant 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. C_LIO_LIThis comprehensive effector profiling revealed that Psa3 carries robust effector redundancy for a large portion of its effectors, covering a few functions critical to disease. C_LI

plant biology↗

bioRxiv2022

The complete genome sequence of Pseudomonas syringae pv. actinidifoliorum ICMP 18803

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).

genomics↗

bioRxiv2022

A lipopolysaccharide-dependent phage infects a pseudomonad phytopathogen and can evolve to evade phage resistance

Bacterial 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 {Phi}Psa374-like phages and show that they use lipopolysaccharides (LPS) as their main receptor. Through proteomics and cryo-electron microscopy of {Phi}Psa374, we revealed the structural proteome and that this phage possess a T=9 capsid triangulation, unusual for myoviruses. Furthermore, we show that {Phi}Psa374 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 {Phi}Psa374. This study provides new insight into the properties of {Phi}Psa374-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.

microbiology↗

bioRxiv2021

Effector loss drives adaptation of Pseudomonas syringae pv. actinidiae to Actinidia arguta

A pandemic isolate of Pseudomonas syringae pv. actinidiae biovar 3 (Psa3) has devastated kiwifruit orchards growing cultivars of Actinidia 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 infected A. arguta leaves, observed by microscopy and quantified by ion-leakage assays. Isolates of Psa3 that cause disease in A. arguta have 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 in A. arguta plantlets than wild-type Psa3. Screening of a complete library of Psa3 effector knockout strains identified increased growth in planta for knockouts of four effectors - AvrRpm1a, HopF1c, HopZ5a, and the EEL effector HopAW1a - suggesting a resistance response in A. 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 in Actinidia for breeding durable resistance into future kiwifruit cultivars.

plant biology↗

bioRxiv2021

A high throughput bispecific antibody discovery pipeline

Bispecific antibodies (BsAbs) represent an emerging class of immunotherapy but inefficiency in the current BsAb discovery paradigm has limited their broad clinical availability. Here we report a high throughput, agnostic, single-cell-based BsAb functional screening pipeline, comprising molecular and cell engineering for efficient generation of BsAb library cells, followed by functional interrogation at the single-cell level to identify and sort positive clones and downstream sequence identification with single-cell PCR and sequencing and functionality characterization. Using a CD19xCD3 bispecific T cell engager (BiTE) as a model system, we demonstrate that our single cell platform possesses a high throughput screening efficiency of up to one and half million variant library cells per run and can isolate rare functional clones at low abundance of 0.008%. Using a complex CD19xCD3 BiTE-expressing cell library with approximately 22,300 unique variants comprising combinatorially varied scFvs, connecting linkers and VL/VH orientations, we have identified 98 unique clones including extremely rare ones ([~] 0.001% abundance). We also discovered BiTEs that exhibit novel properties contradictory to conventional wisdom, including harboring rigid scFv connecting peptide linkers yet with in vitro cytotoxicity comparable to that of clinically approved Blinatumomab. Through sequencing analyses on sorted BiTE clones, we discovered multiple design variable preferences for functionality including the CD19VL-VH- CD3VH-VL and CD19VH-VL-CD3VH-VL arrangements being the most favored orientation. Sequence analysis further interrogated the sequence composition of the CDRH3 domain in scFvs and identified amino acid residues conserved for function. We expect our single cell platform to not only significantly increase the development speed of high quality of new BsAb therapeutics for cancer and other disorders, but also enable identifying generalizable design principles for new BsAbs and other immunotherapeutics based on an in-depth understanding of the inter-relationships between sequence, structure, and function.

bioengineering↗

bioRxiv2020

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

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 characterised 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 pseudogenised. We have shown that HopM1 does not contribute to Psa3 virulence due to a truncation in shcM, a truncation conserved in the Psa lineage, likely due to the need to evade HopM1-triggered immunity in kiwifruit. We characterised 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.

pathology↗

bioRxiv2020

Variation at the common polysaccharide antigen locus drives lipopolysaccharide diversity within the P. syringae species complex

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.

microbiology↗