China
March 14, 2016
Verticillium dahliae is a soil-borne fungal pathogen that has broad host range. In China, it is a causal agent of cotton vascular wilt disease that causes large yield loss due to the lack of efficient controlling methods. Dr. GUO Huishan’s group in the State Key Laboratory of Plant Genomics, Institute of Microbiology of Chinese Academy of Sciences, applied host-induced gene silencing (HIGS) approach in cotton to defend against V. dahliae.
RNA silencing is a conserved regulatory mechanism of gene expression that has been widely characterized in eukaryotic organisms. The basic silencing machinery consists in processing dsRNA precursors into siRNAs to guide selective destruction of target mRNAs.
HIGS has been applied as an effective strategy to defend several pathogenic microbes including fungi. However, there is no experimental evidence of inducing target gene silencing in fungal cells, neither such HIGS in crop protection to natural infection has been assessed.
In cotton that grows in V. dahliae microsclerotia-containing soil, the host-pathogen interaction persists throughout the entire growth period. Therefore, the application of HIGS in cotton to defend against V. dahliae is full of challenge and mystery.
Dr. GUO’s group generated transgenic cotton plants expressing an RNAi construct specifically targets VdH1gene, which had shown to be involved in V. dahliae virulence on cotton. The transgenic cotton indeed shows resistance to V. dahliae, and the siVdH1 signals are detectable in the V. dahliae colonies isolated from the infected transgenic cotton plants.
Furthermore, the isolated V. dahliae colonies show significantly reduced VdH1 gene expression. This work demonstrates for the first time that host-derived small RNAs are able to enter fungal cells during plant-V. dahliae interaction, and induced RNA silencing of the target fungal genes.
The stable transgenic cotton line has also tested for wilt disease resistance in the field of Xinjiang Province of China, and indeed shown enhanced resistance in the V. dahliae distributed area. This achievement provides new strategy and future direction of crop protection against vascular wilt diseases.
The paper entitled “Host-induced gene silencing of the target gene in fungal cells confers effective resistance to the cotton wilt disease pathogen Verticillium dahliae” has been published online in Molecular Plant with the Ph.D. student ZHANG Tao as the first author and Dr. GUO as corresponding author.
Dr. GUO’s group collaborated with Dr. ZHANG Jie’s group and achieved in optimizing the V. dahliae gene knock-out method. Since with the assembly of V. dahliae genome sequences, an increasing number of genes need to be functionally analyzed and annotated which promotes gene targets selection for HISG as well as facilitates V. dahliae functional genomics. However, the traditional V. dahliae gene knock-out method based on homologous recombination relies on the process of constructing a gene-deletion vector and screening the transformants.
To speed up this critical process, the research groups constructed a new vector USER-ATMT-DS for high efficient gene targeting in V. dahliae. This vector has combined all the advantages of vectors used in fungal gene study. Compared to the traditional method, the new vector significantly reduced the time of vector construction and increased the ratio of precise gene targeting.
The application of this vector will highly accelerate the steps of functional genomics in fungi. The data has been published online in Phytopathology with Ph.D. student WANG Sheng as the first author and Dr. GUO and Dr. ZHANG as joint corresponding authors.
Figure: 35S-VdH1i transgenic cotton plants induce silencing of the fungal mRNA and confer resistance to V. dahliae infection. (Image by Dr. GUO’s group)
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