Histocytology And Transcriptomics Studies On Infection Of Malus Domestica Cv. Fuji By Valsa Mali

Apple valsa canker, caused by the fungus Valsa mali Mayabe et Yamada, anamorph: Cytospora sacculus [Schwein.] Gvrit., is one of the major diseases of apple in China. However, limited knowledge exists about the infection process and the mechanisms of the fungal pathogenicity, especially the development of the pathogen in host tissues and the genes involved in fungal pathogenesis, which will impede to develop efficient management strategies to control the apple valsa canker in apple. Thus, the objectives of the current study were (1) to elucidate the behavior and infection processes including conidia germination, pen-etration and spread in host tissues of V. mali using light, scanning and transmission electron microscopy.(2) Enzyme-and immune-gold labeling was conducted to detect the cell wall components alteration.(3) Based on the transcriptome analysis to explicited genes involved in the fungal pathogenesis.(4) Knockout the pectinase gene from the V. mali genome based on the PEG mediated transformation to detect the function of pectinase during the fungal infection.1, the infection process of V. mali, the causal agent of apple canker, in apple (Malus domestica cv. Fuji) tissue was investigated by light and electron microscopy. The germination pattern of conidia was sim-ilar on wounded or intact twigs, but there was a time delay on intact bark. On intact bark tissue, conidia swell-ing could be observed at16h post inoculation (hpi) and conidia germinated at20hpi, however, no infection was observed. And the germinaton ratio was significantly difference on wounded and intact bark,87%of the conidia were germinated on wounded bark while24.7%of the conidia were germinated on the intact bark. On the wounded twig surface, conidia swelled at6hpi, and germinated at16hpi. Direct penetration of germ tubes was observed by20hpi and the pathogen caused canker lesions4days post inoculation (dpi) on wounded twigs and colonized in cortex and phelome tissues. By15dpi, hyphae colonized all bark tissues and xylem vessels and generated pycnidia on the canker region under epidermal. Electron microscopy observation of the canker region showed that fungal hyphae mainly spread intercellularly causing severe tissue maceration and cell necrosis. Fungal colonization resulted in marked alterations in host tissues including plasmolysis and degeneration of protoplasts and cell walls.2, to illustration the function of cell wall enzymes produced by V. mali, cytochemical labeling was conducted by the enzyme-and immuno-gold of cell wall components of the infected tissues. Cellulase and xylanse-gold labeling showed that the density of gold particles of cellulose and xylan was difference insignificantly. However, the pectin over the infected cell wall showed a significant increase in gold particles density. The results indicated that pectinases were involved in pathogenesis of V. mali of apple tissue rather than the cellulase or xylanase.3, a better understanding of this host-pathogen interaction is urgently needed to improve management strategies. In the current study we sequenced the transcriptomes of V. mali during infection of apple bark and mycelium grown in axenic culture using Illumina RNA-Seq technology. A total of66,982contigs were assembled and resulted in13,046genes. We identified437genes that were differentially expressed during fungal infection compared to fungal mycelium grown in axenic culture. One hundred and thirty nine of these437genes showed more than three fold higher transcript abundance during infection. Functional categorization of these139genes revealed that most of them were related to cell wall hydrolysis and secondary metabolite biosynthesis. GO and KEGG enrichment analyses of the induced genes suggest prevalence of genes associated with pectin catabolic, hydrolase activity and secondary metabolite biosynthesis during fungal infection. Our results indicate that cell wall degradation accompanied by cell necrosis-probably caused by secondary metabolites-is essential for disease establishment. Furthermore,585putative pathogenicity determinants and215candidate effectors in V. mali were identified. Additionally,15different expressed genes were chosen for qRT-PCR verification and the results were consistent to FPKM analysis.4, based on the transcriptome seuqneces, two pectinase genes, m.12056(Vmpg-2) and m.13113(Vmpg-3), were chosen for gene function verification. Double-joint PCR was used for the gene knockout cassetes development, and the PEG mediated transformation was used for the pectinase genes knockout from the V. mali genome. Unfortunately, there was no transformant abtained from Vmpg-2deletion. And a total of152transformants were collected from Vmpg-3deletion. Screening the Vmpg-3deletion transformants on the PDA medium that contain100μg/mL hygromycin, PCR applification and the southern blot verification, a mutant of Vmpg-3gene deletion was obtained. Bioassay of the Vmpg-3deletion mutant on the exised leaves was performed and the results showed that the pathogenicity of the Vmpg-3delection mutant decreased but the difference was insignificantly compared with the wild type. The pectinase gene deletion indicated that Vmpg-3affected the pathogenicity of V. mali but really not the determinated factor.