| 研究実績の概要 |
This study has two aims: (1)Elucidate the evolution of genes encoding potential virulence factors, known as effectors, that are organized in clusters with uneven phylogenetic distribution in different species of the Colletotrichum gloeosporioides species complex (CGSC accessory regions). (2)To characterize one of the effectors in this cluster, CfNudix
Aim 1
Chromosome-level assemblies of CGSC strains were generated and analyzed. Enrichment analyses showed CGSC accessory regions are associated with subtelomeres and repeat-rich minichromosomes. Copies of the effector cluster were identified in these regions with related effector genes flanking these clusters. Transcriptional analyses revealed that clustered effectors have a similar expression patterns. Phylogenetic analyses showed that CGSC accessory genes from different species have a common origin but that accessory clusters are independently lost/gained. We obtained 52 additional CGSC strains from global fungal repositories, isolated DNA, prepared libraries, and sequenced these strains.
Aim 2
CfNudix proteins and selected homologs were expressed, purified, and tested on a panel of substrates. Unlike previously identified nudix effectors, CfNudix does not degrade ADP-ribose or NADH, indicating that it has a novel function.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
2: おおむね順調に進展している
理由
(Aim 1) In the proposal, we planned to generate high-quality genome assemblies of 3 different CGSC species using PacBio long reads and analyze them for insights into the evolution of virulence-related genes in 2019 and this is complete. We originally planned to generate RNAseq reads of C. siamense and C. aenigma, but, given that the virulence genes of interest are highly identical and different loci cannot be distinguished by RNAseq, we opted to perform qPCR for transcriptional analyses.
We obtained 52 additional CGSC strains from diverse geographic locations and have started assessing their virulence phenotype on F. vesca. These genomes have been sequenced and the analysis of these genomes is being performed. This work was planned for FY2019-2021 and was on track.
(Aim 2)
We expressed and purified CfNudix and additional homologs and tested them for their substrate specificity using the malachite green assay. However, while we showed that CfNudix does not hydrolyze a variety of targets, we have not yet identified its substrate. This was planned for FY2019-2020 and was on track.
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| 今後の研究の推進方策 |
With the nCov-2 situation, experiments have been disrupted.
(Aim 1)
The sequences of the 52 additional strains are currently being analyzed. 23 strains have been phenotyped. When conditions allow, further phenotyping of the additional strains will be performed. In the meantime, we are generating labelled data to train an automated lesion detection program using the already gathered data which will speed up future phenotyping analyses.
(Aim 2)
We planned to delete CfNudix from our model strain. From the genome analysis, we identified six identical copies of this gene in our model strain. In addition, we found the region flanking this gene is also highly identical, making it challenging to design deletion constructs and screen targeted mutations. This means 6 sequential knock-outs need to be done.
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