基于组学关联分析香蕉枯萎病菌厚垣孢子的形成机制

Fusarium oxysporum f. sp. cubense (FOC) is an important soil-borne fungal pathogen causing banana fusarium wilt, threatens banana production worldwide. The FOC produced three types of spores, including chlamydospores, macroconidia and microconidia. among them, chlamydospores could survival up to 30 years in infected soil and spread through a variety of ways, including diseased seed pieces, soil, irrigation water, farming tools and movement of human and animals. Heretofore, there is no few options to control this disease and formation mechanisms of chlamydospores are largely unknown. To uncover mechanisms of chlamydospores formation, an integrated analysis of transcriptome and metabolomics was conducted to search candidate differential expression genes associated with chlamydopsores formation in FOC. The experimental data revealed that mechanisms of chlamydospores formation are associated with amino sugar and nucleotide sugar metabolism, folate biosynthesis and thiamine metabolism pathways. Through target gene deletion and complementary experiment, the functions of 15 target genes will be determined. Through investigation of chlamydospores formation and structure of cell wall in chlamydospores, and viability of chlamydospores against heat or cold or salt or acid or alkali, we will reveal the relationship between the 3-4 target genes and chlamydospores formation. Using GFP tagging, we will study the localization of proteins encoding by the 3-4 target genes in cell. Moreover, we will design a type of double-stranded RNA (dsRNA) based on coding region of 1-2 targeted genes associated with chlamydospores formation and then assay the effect of the dsRNA against chlamydospores formation. These data will provide theoretical guidance and technical support for integrated control of banana fusarium wilt.

香蕉枯萎病菌是威胁香蕉生产的重要土传病原真菌，其厚垣孢子可在土壤中存活至 30 年，是防控香蕉枯萎病的主要障碍，故所引起的香蕉枯萎病的防控难度极大。为解析该病原菌厚垣孢子的形成机制，本项目对该病原菌厚垣孢子形成前后的转录组和代谢组进行关联分析及氨基糖添加实验验证，揭示厚垣孢子其与氨基糖和核苷酸糖代谢、叶酸合成及硫胺素代谢通路相关；然后，将利用基因敲除和互补技术，获得相关代谢通路 15 个基因的敲除突变体和互补转化子；并对控制厚垣孢子形成的 3-4 个关键基因敲除突变体和互补转化子的厚垣孢子形成情况、细胞壁的结构变化、存活能力及其与致病力的关系和靶基因编码蛋白的亚细胞定位进行分析，阐明该关键基因的作用机制；最后，尝试基于控制厚垣孢子形成的 1-2 个关键基因制备双链 RNA，室内检测其对厚垣孢子形成的抑制作用，探索研发其为新型杀菌剂的可行性。进而为香蕉枯萎病的绿色防控提供理论指导和技术支持。

香蕉枯萎病菌是威胁香蕉生产的重要土传病原真菌，其厚垣孢子可在土壤中存活至 30 年，是防控香蕉枯萎病的主要障碍。解析该病原菌厚垣孢子的形成机制，从控制该病害的最初侵染源的角度来防控香蕉枯萎病是一种新探索。本项目建立了香蕉枯萎病菌厚垣孢子诱导形成体系，研究发现碳源缺乏或者浓度较低时可促进厚垣孢子形成。对该病原菌厚垣孢子形成前后样品开展RNA-seq和代谢组关联分析及N-乙酰葡糖胺、甘氨酸添加实验验证，揭示厚垣孢子形成与氨基糖和核苷酸糖代谢通路、氨基酸合成通路相关。利用同源重组策略，获得了相关代谢通路的基因敲除突变体。与野生型菌株相比，ΔFoNagA的厚垣孢子和分生孢子产量显著减少，ΔFoRlm1 的分生孢子产量显著减少，但厚垣孢子产量无显著变化；该2个突变体的厚垣孢子经高温处理后，萌发率显著降低。致病性测试表明ΔFoNagA和ΔFoRlm1的厚垣孢子均对香蕉苗的致病性减弱。透射电镜检测表明ΔFoNagA 和ΔFoRlm1厚垣孢子的细胞壁显著变薄。平板检测表明ΔFoNagA和ΔFoRlm1 气生菌丝均显著减少，且ΔFoRlm1 在 YPD 中的菌丝干重显著降低。氧化应激测试表明ΔFoNagA和ΔFoRlm1 均对过氧化氢过敏感。实时荧光定量PCR表明FoNagA和FoRlm1的敲除均影响了香蕉枯萎病菌镰孢菌酸和白僵菌素合成基因的转录。对该病菌野生型菌株和突变体 ΔFoRlm1的转录组进行了比较，KEGG富集揭示DEGs主要富集在氨基酸合成通路。上述结果说明，FoNagA和FoRlm1的敲除影响了香蕉枯萎病菌的生理特性，从而影响厚垣孢子的耐受能力及致病性。本项目基于FoNagA制备双链 RNA，室内检测表明其对厚垣孢子形成有明显的抑制作用，由此说明，其具有发展为新型双链RNA杀菌剂的潜力。进而为香蕉枯萎病的绿色防控提供理论指导和技术支持。