Genome‐wide identification of pathogenicity genes in Xanthomonas oryzae pv. oryzae by transposon mutagenesis

A transposon mutant library was constructed from the bacterial blight pathogen Xanthomonas oryzae pv. oryzae (Xoo) KACC10331 by Tn5 transposon mutagenesis. The susceptible rice cultivar Milyang 23 was inoculated with a total of 24 540 mutants resistant to kanamycin and 67 avirulent or reduced‐pathogenicity mutant strains were selected for study. Southern hybridization verified that 84 mutant strains had single‐copy insertions and their single‐transposon insertion sites were identified by sequencing analysis combined with thermal asymmetric interlaced (TAIL)‐PCR. The single‐transposon‐tagged sequences of 21 mutant strains belonged to pathogenicity‐related genes previously reported in Xanthomonas species, while the other 46 single‐transposon‐tagged sequences included diverse functional genes encoding, five cell‐wall‐degrading enzymes, three fimbrial and flagella assembly regulators, five regulatory proteins, 15 metabolic regulators and 18 hypothetical proteins, which were identified as novel pathogenicity genes of Xoo.     

Cotyledons are the main source of secondary spread of Acidovorax citrulli in melon nurseries

Acidovorax citrulli (Ac) is the causal agent of bacterial fruit blotch (BFB) disease resulting in substantial economic damage to cucurbit crops worldwide. The plant parts and cultural practices (irrigation methods and bactericidal sprays) that affect the secondary spread of Ac in melon nurseries were investigated in this study. Overhead irrigation dispersed the pathogen from infected seedlings to 95% of the neighbouring healthy seedlings, with 80% of them displaying high disease severity. In contrast, when sub‐irrigation by floating was employed, the neighbouring plants of the infected ones did not display disease symptoms and were not colonized by Ac. Foliar treatment with Kocide after cotyledon emergence reduced disease incidence to 40%, with 37% of the plants displaying low disease severity. Assessment of Ac populations in different parts of the seedlings revealed that cotyledons were the most colonized part of the plant. Images of fluorescent binocular and confocal laser‐scanning microscopy of seedlings infected with a GFP‐labelled Ac strain showed that the pathogen forms abundant aggregates on the surface of cotyledons, colonizes the intercellular spaces of the parenchymatic tissues extensively, and moves through the vascular system of the hypocotyls, leading to infection of emerging leaves. Results of this study indicate that preventing secondary spread of Ac in melon nurseries by sub‐irrigation combined with a bactericidal spray at the cotyledon stage may provide an effective means for BFB control.     

Long‐term survival of Acidovorax citrulli in citron melon (Citrullus lanatus var. citroides) seeds

Long‐term survival of Acidovorax citrulli in citron melon (Citrullus lanatus var. citroides) seeds was investigated. Citron melon seed lots infected with A. citrulli were generated in the field by inoculating either the pistils (stigma) or pericarps (ovary wall) of the female blossoms. Seventeen A. citrulli isolates from 14 different haplotypes belonging to two different groups (group I and II) were used for inoculation. After confirming that 100% of seed lots were infected, they were stored at 4°C and 50% RH for 7 years. After storage, the viability of A. citrulli cells from individual lots was determined by plating macerated seeds on semiselective medium as well as growing seeds for 14 days and scoring for bacterial fruit blotch symptoms. The type of A. citrulli isolate (group I or group II) used did not significantly influence bacterial survival. However, A. citrulli survival was significantly greater in seed lots generated via pistil inoculation (52·9 and 29·4%) than via pericarp inoculation (23·5 and 17·6%). Repetitive extragenic palindrome (rep)‐PCR on A. citrulli isolated from citron melon seed lots after storage displayed similar fingerprinting patterns to those of the reference strains originally used for blossom inoculation, indicating that cross‐contamination did not occur. The results indicate that A. citrulli may survive/overwinter in citron melon seeds for at least 7 years and bacterial survival in seed was influenced more by method of blossom inoculation than by the type of bacterial isolate.     

西甜瓜果斑病菌avrRxo1同源基因激发玉米Rxo1抗性产生的研究

生物信息学分析发现西(甜)瓜果斑病菌(Acidovorax citrulli,Ac)基因组中存在水稻条斑病菌(Xanthomonas oryzae pv.oryzicola,Xoc)avrRxo1的同源基因avrRxo1_(Ac)。为了揭示avrRxo1_(Ac)是否能够激发玉米R基因Rxo1介导的过敏反应(Hypersensitive response,HR),本研究克隆了avrRxo1_(Ac)基因;氨基酸同源性分析结果显示:AvrRxo1_(Ac)与AvrRxo1的同源性为53.16%。将avrRxo1_(Ac)置于Xoc的avrRxo1突变体中,发现其不能恢复该突变体在含有Rxo1玉米上激发HR反应的能力;借助农杆菌瞬时表达体系发现AvrRxo1A,也不能在Rxo1玉米上激发HR反应。这些结果表明avrRxo1_(Ac)-Rxo1互作不表现为基因-对-基因关系,暗示Rxo1抗病基因转入西甜瓜中,将不能起到抗西(甜)瓜细菌性果斑病的作用。     

水稻条斑病菌hrpB1基因决定寄主致病性和非寄主过敏反应的功能研究

水稻条斑病菌(Xanthomonas oryzaepv.oryzicola,Xoc)的hrp基因决定了病原菌在非寄主植物上的过敏反应(hypersensitive response,HR)和在寄主植物上的致病性(pathogenicity),基因产物形成Ⅲ型分泌系统(type-Ⅲ secretion system,T3SS)将致病性效应分子注入寄主细胞从而引起水稻产生抗病性或者感病性反应。以位于hrpB操纵单元的首个hr-pB1基因为对象,通过基因敲除方式对其进行了突变,发现hrpB1突变体丧失了在水稻上的致病性和在烟草上激发HR的能力,并且在水稻组织中的生长能力显著降低。RT-PCR测定结果表明,hrpB1的转录表达受HrpG和HrpX的正调控。免疫杂交结果显示,HrpB1蛋白可通过T3SS进行分泌。这些结果不仅明确了hrpB1基因在病原菌致病性中的功能,而且提示了hrp结构基因不仅仅局限于形成Ⅲ型分泌系统,部分hrp基因产物本身也通过Ⅲ型系统分泌到胞外,并且可能起到效应分子的功能。     

猕猴桃溃疡病菌不致病菌株G230的鉴定及形成原因分析

为探索田间猕猴桃溃疡病菌Pseudomonas syringae pv. actinidiae(Psa)致病力丧失的分子机制,针对从猕猴桃果园中分离获得的1株不致病菌株G230,通过特异性引物检测和多基因序列分析明确其分类地位,并设计引物检测其是否由已知遗传变异引起,通过比较基因组学、基因表达、超敏反应和荧光素酶报告菌株检测确定引起菌株G230致病力丧失的原因。结果表明,不致病菌株G230为Psa生物型3(Psa3),其致病缺陷不是由已报道的遗传变异引起;基于基因组比较分析发现菌株G230中的hrpS基因被转座子ISPsy36插入破坏,导致Ⅲ型分泌系统（type Ⅲ secretion system,T3SS）不能正常表达;而在不致病菌株G230中表达hrpS基因后能恢复其T3SS功能,使其具备致病能力及激发非寄主超敏反应的能力。表明转座子ISPsy36插入hrpS基因内部可以破坏Psa的T3SS功能进而使其丧失致病力,这是自然条件下Psa3丧失致病力的一种新型机制。