Abstract Fungal plant-pathogens promote infection of their hosts through the release ‘effectors’—a broad class cytotoxic or virulence-promoting molecules. Effectors may be recognised by resistance sensitivity receptors in host, which can determine disease outcomes. Accurate prediction effectors remains a major challenge plant pathology, but if achieved will facilitate rapid improvements to host resistance. This study presents novel tool and pipeline for ranking predicted effector...

Ascochyta rabiei is the causal organism of ascochyta blight chickpea and present in crops worldwide. Here we report release a high-quality PacBio genome assembly for Australian A. isolate ArME14. We compare ArME14 with an Illumina Indian isolate, ArD2. The has gapless sequences nine chromosomes telomere at both ends 13 large contig that extend to one telomere. total length was 40,927,385 bp, which 6.26 Mb longer than ArD2 assembly. Division by OcculterCut into GC-balanced AT-dominant...

Chocolate spot is a major fungal disease of faba bean caused by the ascomycete fungus, Botrytis fabae. B. fabae also implicated in botrytis gray mold lentils, along with cinerea. Here we have isolated and characterized two isolates from chocolate lesions on leaves. In plant assays lentil, was more aggressive than cinerea observed variation susceptibility among small set cultivars for both hosts. Using light microscopy, spreading, generalized necrosis response toward contrast, to localized...

Ascochyta lentis, the causal organism of blight (AB) lentil (Lens culinaris), has been shown to produce an avirulence effector protein that mediates AB resistance in certain cultivars. The two known forms were identified from a biparental mapping population between isolates have reciprocal virulence on 'PBA Hurricane XT' and 'Nipper'. AlAvr1-1 was described for PBA XT-avirulent isolate P94-24 AlAvr1-2 characterized XT-virulent AlKewell. Here, we performed genome-wide association study...

Modified pEAQ-HT-DEST1 vectors were used for agroinfiltration in legumes. We demonstrate protein expression and export pea, lentil, faba bean; however, the method chickpea was not successful. Agroinfiltration is a valuable research investigating virulence avirulence effector proteins from pathogens pests, where heterologous are transiently expressed plant leaves hypersensitive necrosis responses other functions can be assessed. Nicotiana benthamiana widely characterisation of broad-spectrum...

Ascochyta lentis causes ascochyta blight in lentil (Lens culinaris Medik.) and yield loss can be as high 50%. With careful agronomic management practices, fungicide use, advances breeding resistant varieties, disease severity impact to farmers have been largely controlled. However, evidence from major producing countries, Canada Australia, suggests that A. isolates change their virulence profile level of aggressiveness over time under different selection pressures. In this paper, we describe...

Chromosomal rearrangements of band 19q13.4 are frequent cytogenetic alterations in benign thyroid adenomas. Apparently, these lead to the upregulation genes encoding microRNAs two clusters mapping breakpoint region, i.e. miR-371-3 and C19MC. Since members both have been associated with neoplastic growth other tumor entities question arises whether or not their predisposes malignant transformation follicular cells thyroid. To address this we quantified expression miR-372 miR-520c-3p samples...

Abstract Ascochyta lentis is a fungal pathogen that causes ascochyta blight in the important grain legume species lentil, but little known about molecular mechanism of disease or host specificity. We employed map‐based cloning approach using biparental A . population to clone gene AlAvr1‐1 encodes avirulence towards lentil cultivar PBA Hurricane XT. The mapping was produced by mating isolate P94‐24, which pathogenic on Nipper and avirulent Hurricane, Al Kewell, not Nipper. Using...

Ascochyta fabae Speg. is a serious foliar fungal disease of faba bean and constraint to production worldwide. This study investigated the phenotypic genotypic diversity A. pathogen population in southern Australia pathogenic variability was examined on differential set cultivars. The host inoculated with 154 isolates collected from 2015 2018 range reactions high low aggressiveness observed. Eighty percent were categorized as pathogenicity group (PG) PG-2 (pathogenic Farah) detected every...

Sustainable crop production is constantly challenged by the rapid evolution of fungal pathogens equipped with an array host infection strategies and survival mechanisms. One devastating that infect lentil ascomycete Ascochyta lentis which causes black spot or ascochyta blight (AB) on all above ground parts plant. In order to explore mechanisms involved in pathogenicity A. lentis, we developed a targeted gene replacement method using Agrobacterium tumefaciens mediated transformation (ATMT)...

The plant immune system is made up of a complex response network that involves several lines defense to fight invading pathogens. Fungal pathogens on the other hand, have evolved range ways infect their host. interaction between Ascochyta lentis and two lentil genotypes was explored investigate progression ascochyta blight (AB) in lentils. In this study, we developed an Agrobacterium tumefaciens-mediated transformation for A. by constructing new binary vector, pATMT-GpdGFP, constitutive...

Adapted from Chi, M. H., Park, S. Y., & Lee, Y. H. (2009). Colony PCR on fungal colonies grown plates does not work as well it for bacteria (it usually doesn't at all). DNA therefore needs to be extracted first. As this will only used a template check presence / absence of individual genes I am too concerend about high molecular weight or purity. This protocol is quick and can toprocess several samples the same time.

Extracting pure high-molecular weight DNA from some fungal species is difficult due to the presence of polysaccharides and potentially other compounds which biochemically mimic or interfere with extraction process. Such can co-elute in many methods, being separate fom DNA. Although contaminant may not be detected by spectrophotometers fluorometric devices, it substantially interferes long-read sequencing, such as Oxford Nanopore Technologies. To partially resolve this, a protocol presented...

This protocol yields about 5 ml of electrocompetent Agrobacterium tumefaciens cells, aliquotted into 80 ul you get 50-60 tubes out it. Rifampicin is dark red and the colour will change over time, it does however NOT interfere with OD600 measurements, so can use LB to blank.

Adapted from Chi, M. H., Park, S. Y., & Lee, Y. H. (2009). Colony PCR on fungal colonies grown plates does not work as well it for bacteria (it usually doesn't at all). DNA therefore needs to be extracted first. As this will only used a template check presence / absence of individual genes I am too concerend about high molecular weight or purity. This protocol is quick and can toprocess several samples the same time.

This is a slightly modified version of Thermo Fisher's gateway BP protocol which uses less enzyme and therefore more economical.

A quick guide on how to electroporate your plasmid of interest into electrocompetent Agrobacterium tumefaciens cells.

This is a slightly modified version of Thermo Fisher's gateway LRprotocol which uses less enzyme and therefore more economical.

GateWay recombination cloning is achieved by flanking your gene of interest with attachment sites. In our case attB1 and attB2. Those sites are added to the PCR product via primers 5' extensions. Since those primes create 31 bp 30 primer extensions respectively, plus about 20 actual binding sequence it becomes expensive fast if you need 2 x ~50 for every GOI. We therefore use a step process attach attB2 first run specific carrying short extesions, then second utilizing universal which bind...

This method uses Agrobacterium tumefaciens to transfer a piece of TDNA containing resistance gene (hph for hygromycin in this particular case) flanked by homologous regions the you want knock out into fungal spore. The process is broken down following steps: Assembly knockout construct via Gibson assembly Transformation E.coli Plasmid harvest and transformation spores Verifying

GateWay recombination cloning is achieved by flanking your gene of interest with attachment sites. In our case attB1 and attB2. Those sites are added to the PCR product via primers 5' extensions. Since those primes create 31 bp 30 primer extensions respectively, plus about 20 actual binding sequence it becomes expensive fast if you need 2 x ~50 for every GOI. We therefore use a step process attach attB2 first run specific carrying short extesions, then second utilizing universal which bind...