Biology subjects

Arshed, S.

Publications and source records attributed to Arshed, S..

4 recordsLinked to original sources

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

The Bcvic1 and Bcvic2 vegetative incompatibility genes in Botrytis cinerea encode proteins with domain architectures involved in allorecognition in other filamentous fungi

Vegetative incompatibility is a fungal allorecognition system characterised by the inability of genetically distinct conspecific fungal strains to form a viable heterokaryon, and is controlled by multiple polymorphic loci termed vic (vegetative incompatibility) or het (heterokaryon incompatibility). We have genetically identified and characterised the first vic locus in the economically important, plant-pathogenic, necrotrophic fungus Botrytis cinerea. A bulked segregant approach coupled with whole genome Illumina sequencing in near-isogenic lines of cinerea was used to map a 60-kb genomic region for a vic locus. Within that locus, we identified two adjacent, highly polymorphic open reading frames, Bcvic1 and Bcvic2, which encode predicted proteins that contain domain architectures implicated in vegetative incompatibility in other filamentous fungi. Bcvic1 encodes a predicted protein containing a putative serine esterase domain, a NACHT family of NTPases domain, and several Ankyrin repeats. Bcvic2 encodes a putative syntaxin protein containing a SNARE domain; such proteins typically function in vesicular transport. Deletion of Bcvic1 and Bcvic2 individually had no effect on vegetative incompatibility. However, deletion of the region containing both Bcvic1 and Bcvic2 resulted in mutant lines that were severely restricted in growth and showed loss of vegetative incompatibility. Complementation of these mutants by ectopic expression restored the growth and vegetative incompatibility phenotype, indicating that Bcvic1 and Bcvic2 are controlling vegetative incompatibility at this vic locus. Author SummaryFungal colonies are characterised by radiating filaments, termed hyphae, which often fuse to form a highly interconnected individual. This is advantageous since it enables efficient water and nutrient utilisation across a colony network. However, hyphal fusion is not necessarily restricted to within an individual colony, with potential for hyphal fusion between individuals belonging to the same species. There are, however, drawbacks to this. For instance, viruses that detrimentally affect a colony may be transmitted, with their infection leading to a reduction in the virulence of a pathogenic species. Fungi have therefore developed complex systems to prevent fusion between genetically distinct individuals of the same species. This phenomenon is termed vegetative incompatibility and results in the death of fused cells and cessation of transfer of cellular contents from one individual to another. We have identified the first genes in the fungal plant pathogen Botrytis cinerea that control this phenomenon. They resemble genes that control vegetative incompatibility in other fungi, and genes involved in immunity in plants and animals. Uncovering further genes involved in vegetative incompatibility in B. cinerea may pave the way for the development of a super donor strain capable of overriding vegetative incompatibility to transmit viruses, thus enabling their exploitation as potent control agents against this damaging plant pathogen.

genetics↗

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

CRISPR-Cas9 gene editing and rapid detection of gene-edited mutants using high-resolution melting in the apple scab fungus, Venturia inaequalis

BackgroundScab, or black spot, caused by the filamentous fungal pathogen Venturia inaequalis, is the most economically important disease of apple (Malus x domestica) worldwide. To develop durable control strategies against this disease, a better understanding of the genetic mechanisms underlying the growth, reproduction, virulence and pathogenicity of V. inaequalis is required. A major bottleneck for the genetic characterization of V. inaequalis is the inability to easily delete or disrupt genes of interest using homologous recombination. Indeed, no gene deletions or disruptions in V. inaequalis have yet been published. Recently, CRISPR-Cas9 has emerged as an efficient tool for gene editing in filamentous fungi. With this in mind, we set out to establish CRISPR-Cas9 as a gene editing tool in V. inaequalis. ResultsWe showed that CRISPR-Cas9 can be used for gene inactivation in the apple scab fungus. As a proof of concept, we targeted the melanin biosynthesis pathway gene trihydroxynaphthalene reductase (THN), which has previously been shown to result in a light-brown colony phenotype when transcriptionally silenced using RNA interference. Using one of two CRISPR-Cas9 single guide RNAs (sgRNAs) targeted to the THN gene, delivered by a single autonomously replicating Golden Gate-compatible plasmid, we were able to identify six of 36 stable transformants with a light-brown phenotype, indicating an ~16.7% gene inactivation efficiency. Notably, of these six THN mutants, five had an independent mutation. As part of our pipeline, we also report a high-resolution melting (HRM) curve protocol for the rapid detection of CRISPR-Cas9 gene-edited mutants of V. inaequalis. This protocol identified a single base pair deletion mutation in a sample containing only 5% mutant genomic DNA, indicating high sensitivity for mutant screening. ConclusionsIn establishing CRISPR-Cas9 as a tool for gene editing in V. inaequalis, we have provided a strong starting point for studies aiming to decipher the function of genes associated with the growth, reproduction, virulence and pathogenicity of this fungus. The associated HRM curve protocol will enable CRISPR-Cas9 transformants to be screened for gene inactivation in a high-throughput and low-cost manner, which will be particularly powerful in cases where the CRISPR-Cas9-mediated gene inactivation efficiency is low.

microbiology↗