Reference genome assembly for Australian Ascochyta lentis isolate Al4

Ascochyta lentis causes ascochyta blight in lentil (Lens culinaris Medik.) and yield loss can be as high as 50%. With careful agronomic management practices, fungicide use, and advances in breeding resistant lentil varieties, disease severity and impact to farmers have been largely controlled. However, evidence from major lentil producing countries, Canada and Australia, suggests that A. lentis isolates can change their virulence profile and level of aggressiveness over time and under different selection pressures. In this paper, we describe the first genome assembly for A. lentis for the Australian isolate Al4, through the integration of data from Illumina and PacBio SMRT sequencing. The Al4 reference genome assembly is almost 42 Mb in size and encodes 11,638 predicted genes. The Al4 genome comprises 21 full-length and gapless chromosomal contigs and two partial chromosome contigs each with one telomere. We predicted 31 secondary metabolite clusters, and 38 putative protein effectors, many of which were classified as having an unknown function. Comparison of A. lentis genome features with the recently published reference assembly for closely related A. rabiei show that genome synteny between these species is highly conserved. However, there are several translocations and inversions of genome sequence. The location of secondary metabolite clusters near transposable element and repeat-rich genomic regions was common for A. lentis as has been reported for other fungal plant pathogens.

Targeted Disruption of Scytalone Dehydratase Gene Using Agrobacterium tumefaciens-Mediated Transformation Leads to Altered Melanin Production in Ascochyta lentis

Sustainable crop production is constantly challenged by the rapid evolution of fungal pathogens equipped with an array of host infection strategies and survival mechanisms. One of the devastating fungal pathogens that infect lentil is the ascomycete Ascochyta lentis which causes black spot or ascochyta blight (AB) on all above ground parts of the plant. In order to explore the mechanisms involved in the pathogenicity of A. lentis, we developed a targeted gene replacement method using Agrobacterium tumefaciens mediated transformation (ATMT) to study and characterize gene function. In this study, we investigated the role of scytalone dehydratase (SCD) in the synthesis of 1,8-dihydroxynaphthalene (DHN)-melanin in AlKewell. Two SCD genes have been identified in AlKewell, AlSCD1 and AlSCD2. Phylogenetic analysis revealed that AlSCD1 clustered with the previously characterized fungal SCDs; thus, AlSCD1 was disrupted using the targeted gene replacement vector, pTAR-hyg-SCD1. The vector was constructed in a single step process using Gibson Assembly, which facilitated an easy and seamless assembly of multiple inserts. The resulting AlKewell scd1::hyg transformants appeared light brown/brownish-pink in contrast to the dark brown pycnidia of the WT strain and ectopic transformant, indicating an altered DHN-melanin production. Disruption of AlSCD1 gene did not result in a change in the virulence profile of AlKewell towards susceptible and resistant lentil varieties. This is the first report of a targeted gene manipulation in A. lentis which serves as a foundation for the functional gene characterization to provide a better understanding of molecular mechanisms involved in pathogen diversity and host specificity.

Transcriptome profiling of lentil in response to Ascochyta lentis infection

Aim of study: The purpose was to identify some general and genotypic-specific defense responses, in order to obtain a set of candidate genes presumably involved in the resistance.Area of study: The experiment was carried out in León, Spain.Material and methods: We have analyzed the response of three lentil genotypes to Ascochyta lentis (isolate AL 84) at transcriptomic level using the Massive Analysis of cDNA Ends (MACE) technique: the susceptible cultivar 'Lupa', the moderately resistant 'ILL5588' and the resistant wild accession 'BG 16880' (L. culinaris subsp. orientalis).Main results: MACE results yielded a total of 50,935 contigs. The average number of detected contigs in each of the six samples was approximately of 40,000. In response to Ascochyta infection, the jasmonic acid pathway and the lignin biosynthesis were up-regulated in resistant genotypes, while they were down-regulated in the susceptible one. The response to chitin, the salicylic pathway and the auxin response were activated only in the resistant L. c. culinaris genotype, while the giberellin synthesis was only induced in the susceptible L. c. culinaris cv. 'Lupa'. A set of 18 lentil gene sequences putatively involved in the response to the pathogen were validated by RT-qPCR.Research highlights: It can be concluded that in response to the infection by Ascochyta, the lignin biosynthesis and the JA pathway were critical for the resistance, while the giberellin synthesis seems to be related with susceptibility to the pathogen.