Sun Wanqing, Zhao Lihong, Zhou Jinglong, Feng Hongjie, Zhang Yalin, Feng Zili, Zhu Heqin, Wei Feng
Zhengzhou Research Base, State Key Laboratory of Cotton Bio-breeding and Integrated Utilization, School of Agricultural Sciences, Zhengzhou University, Zhengzhou, China.
State Key Laboratory of Cotton Bio-breeding and Integrated Utilization, Institute of Cotton Research of Chinese Academy of Agricultural Sciences, Anyang, Henan, China.
Front Microbiol. 2024 Jul 24;15:1429755. doi: 10.3389/fmicb.2024.1429755. eCollection 2024.
, a soil-borne fungal pathogen, can cause cotton Verticillium wilt. In this study, VdP5CDH, the member of the ALDH_F4-17 family of carboxylate dehydrogenases, was identified in the genome of and investigated function in regulating virulence by generating gene deletion mutants and complementary mutants.
Homologous recombination method was used to construct mutants, transcriptome sequencing revealed gene-related metabolic pathways, and disease degree of cotton was observed through pathogen infection experiments.
The conidial surface of deletion strains was dented and shriveled, and the number of conidial spores increased. Compared with the wild-type (WT), the mycelial diameter of deletion mutants increased by 10.59%-11.16%, the mycelial growth showed irregular branching patterns, and misaligned arrangement. Although capable of penetrating cellophane, deletion mutants were unable to produce melanin. was mainly associated with glucose metabolism, nitrogen metabolism, ABC transporter activity as well as various amino acid metabolic processes. After gene knockout, raffinose and pectin were used as the main carbon sources to promote the growth of strains and the growth rate of deletion strains in the medium containing raffinose was higher than that of WT. Consequently, the deletion mutant strains decreased utilization efficiency with which they utilized various nitrogen sources. The deletion mutants maintain responsiveness to osmotic stress and oxidative stress stimuli. Additionally, compared to WT strains, the deletion mutant strains exhibited differences in culture temperature tolerance, UV exposure response, and fungicide sensitivity. After cotton was infected with deletion strains conidial suspension, its disease index increased dramatically, while it gradually decreased after spraying with 2 mM glutamate in batches. With the increase of spraying times, the effect was more significant, and the disease index decreased by 18.95%-19.66% at 26 dpi.
These results indicated that regulates the pathogenicity of fungi and controls mycelia growth, melanin formation, conidia morphology, abiotic stress resistance, and the expression of infecting structure-related genes.
黄萎病菌(Verticillium dahliae)是一种土传真菌病原体,可引起棉花黄萎病。在本研究中,在黄萎病菌基因组中鉴定出羧酸脱氢酶ALDH_F4 - 17家族成员VdP5CDH,并通过构建基因缺失突变体和互补突变体研究其在调节毒力方面的功能。
采用同源重组方法构建突变体,转录组测序揭示基因相关代谢途径,并通过病原菌感染实验观察棉花的病情程度。
缺失菌株的分生孢子表面凹陷、皱缩,分生孢子数量增加。与野生型(WT)相比,缺失突变体的菌丝直径增加了10.59% - 11.16%,菌丝生长呈现不规则分支模式且排列不齐。缺失突变体虽然能够穿透玻璃纸,但无法产生黑色素。VdP5CDH主要与葡萄糖代谢、氮代谢、ABC转运蛋白活性以及各种氨基酸代谢过程相关。基因敲除后,棉子糖和果胶作为主要碳源促进菌株生长,且缺失菌株在含棉子糖培养基中的生长速率高于WT。因此,缺失突变体菌株降低了利用各种氮源的效率。缺失突变体保持对渗透胁迫和氧化胁迫刺激的响应能力。此外,与WT菌株相比,缺失突变体菌株在培养温度耐受性、紫外线照射响应和杀菌剂敏感性方面表现出差异。棉花接种缺失菌株分生孢子悬浮液后,病情指数显著增加,而分批喷施2 mM谷氨酸后病情指数逐渐下降。随着喷施次数增加,效果更显著,在接种后26天病情指数下降了18.95% - 19.66%。
这些结果表明VdP5CDH调节真菌的致病性,并控制菌丝生长、黑色素形成、分生孢子形态、非生物胁迫抗性以及感染结构相关基因的表达。