糖基转移酶基因rbfCxoo缺失突变导致水稻白叶枯病菌毒性表达增强

摘 要：【目的】阐明水稻白叶枯病菌(Xanthomonas oryzae pv.oryzae,简称Xoo)基因组中推导的脂多糖O抗原合成蛋白基因rbfCxoo (XOO2599) 的结构和生物学功能。【方法】通过基因克隆、序列分析、缺失突变和表型测定,对rbfCxoo的分子特征及其功能进行了鉴定。【结果】 用特异性引物进行PCR扩增,从野生型菌株PXO99A基因组DNA中成功地获得了与己测序菌株KACC10331序列完全一致的全长基因序列; RbfCxoo序列N端和C端分别具有一个糖基转移酶的保守结构域(Glycos_transf_2)。用标记置换法获得了基因缺失突变体△rbfCxoo。与PXO99A相比,△rbfCxoo 脂多糖O抗原合成能力并未发生变化,但鞭毛素糖基化能力有所降低。此外,△rbfCxoo鞭毛运动性、生物膜形成和胞外纤维素酶和木聚糖酶活性都无明显改变,但对水稻的致病性显著增强,毒性相关基因的表达也有所增加。【结论】RbfCxoo可能与细菌鞭毛素糖基化修饰以及毒性表达有关。     

Involvement of Gluconeogenic Pathway in Virulence of Xanthomonas oryzae pv. oryzae

Xanthomonas oryzae pv. oryzae causes bacterial leaf blight, one of the most widespread and destructive bacterial diseases of rice. A phosphoenolpyruvate synthase (ppsA)‐disrupted mutant OSPAM was generated by homologous suicide plasmid integration. The mutant was unable to grow in medium with pyruvate or C₄‐dicarboxylates as the sole carbon source, compared with the wild‐type, indicating a disruption in ppsA function. The mutant showed a reduction in virulence on rice but still induced a hypersensitive response in tobacco. When the mutant was complemented, the response was recovered to wild‐type. These results suggested that X. oryzae pv. oryzae possesses only PPSA route in gluconeogenesis, which is necessary for virulence.     

hrpD6基因决定白叶枯病菌在烟草上的过敏反应和在水稻上的致病性

【目的】白叶枯病菌hrp基因簇由包括hrpD6在内的26个hpa-hrp-hrc基因组成,与植物互作后形成Ⅲ型分泌系统(T3S),将T3S效应分子注入寄主细胞中从而决定在非寄主上的过敏反应(HR)和在水稻上的致病性。但hrpD6基因是否参与了白叶枯病菌在非寄主上的过敏反应(HR)和在水稻上的致病性(pathogenicity)还不清楚。【方法】借助同源重组方法,本研究对白叶枯病菌hrpD6基因进行了突变。【结果】PCR和Southern杂交结果显示,hrpD6基因被成功敲除。烟草上测定结果显示,hrpD6突变体ΔPhrpD6丧失了HR激发能力。致病性测定发现,ΔPhrpD6在水稻苗期不能形成水渍症状,在成株期水稻上不具有致病性,并且细菌生长能力显著下降。功能互补结果显示,hrpD6基因可恢复ΔPhrpD6在烟草上激发HR和在水稻上的致病性以及在水稻组织中的生长能力。RT-PCR结果显示,hrpD6基因的转录表达不仅受水稻诱导,而且受hrpG和hrpX基因调控。不仅如此,hrpD6基因突变还影响T3S效应分子hpa1基因的转录表达和Hpa1蛋白的分泌,暗示hrpD6基因对hpa1基因转录表达具有调控作用。【结论】hrpD6基因的缺失导致白叶枯病菌不能激发烟草产生HR和和丧失在水稻上的致病性,主要是HrpD6对hpa1基因转录表达具有调控作用,并影响T3S效应分子Hpa1的分泌。这些结果为进一步分析hrpD6是否参与T3S分泌装置的形成和调控其它hrp基因的转录表达从而决定病菌在非寄主上的HR和在水稻上的致病性,提供了科学线索。     

水稻黄单胞菌hrp基因簇中致病相关基因hpaB的鉴定水稻黄单胞菌 TAIL-PCR hrp基因簇 hpaB基因

由水稻黄单胞菌引起的水稻白叶枯病是水稻最严重的细菌性病害.通过筛选18000个Xoo Tn5转座子插入突变体,得到其中一个致病力缺失的突变体XOG11.TAIL-PCR方法分离该突变体中插入转座子的侧翼序列,发现转座子插入到位于hrp基因簇的hpaB基因中.对该基因进一步的分析表明该基因编码一个含有156个氨基酸,等电点为4.28,亮氨酸含量为14.4%的蛋白HpaB.Southern blot和PCR验证表明Tn5在该突变体中为单拷贝插入且未发生转座子携带侧翼序列的转移.将hpaB克隆到具有广泛寄主的质粒pHM1中,转化重组质粒进入突变体后,突变体恢复了在其寄主水稻IR24上的致病力,而转化空质粒pHM1后的突变体仍然表现为致病力缺失.证实了水稻黄单胞菌中hpaB基因与该细菌的致病力相关,在侵染水稻的过程中起着不可缺失的作用.     

水稻条斑病菌胞外多糖相关基因的鉴定

摘要:【目的】前期研究中从Tn5 转座子插入的水稻条斑病菌突变体库中获得了17 个胞外多糖改变的突变体。【方法】本文对这些突变体的Tn5 插入位点和基因类型进行了鉴定。【结果】结果显示,胞外多糖减少的11 个突变体中多数为Tn5 插入在已知的gum、xan 和wxoc 基因簇上,Xoryp_4217、Xoryp_2488 和Xoryp_0918为未知的与胞外多糖产生有关的基因,属首次报道;6 个胞外多糖增多的突变体中,fimO、pilY 和xopQ 与胞外多糖产生有关,但在水稻条斑病菌中未见报道;Xoryp2392、Xoryp_4221 和Xoryp_3511 为首次鉴定,其中Xoryp_3511 仅在水稻黄单胞菌中存在。毒性测定结果显示,胞外多糖减少的突变体在水稻上的毒性变弱,而胞外多糖增加的突变体在水稻上的毒性没有显著变化。【结论】这些结果为进一步分析水稻条斑病菌胞外多糖代谢途径以及与水稻的互作关系奠定了基础。     

A mutation in an exoglucanase of Xanthomonas oryzae pv. oryzae,which confers an endo mode of activity,affects bacterial virulence,but not the induction of immune responses,in rice

Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial blight, a serious disease of rice. Xoo secretes a repertoire of cell wall‐degrading enzymes, including cellulases, xylanases and pectinases, to degrade various polysaccharide components of the rice cell wall. A secreted Xoo cellulase, CbsA, is not only a key virulence factor of Xoo, but is also a potent inducer of innate immune responses of rice. In this study, we solved the crystal structure of the catalytic domain of the CbsA protein to a resolution of 1.86 Å. The core structure of CbsA shows a central distorted TIM barrel made up of eight β strands with N‐ and C‐terminal loops enclosing the active site, which is a characteristic structural feature of an exoglucanase. The aspartic acid at the 131st position of CbsA was predicted to be important for catalysis and was therefore mutated to alanine to study its role in the catalysis and biological functions of CbsA. Intriguingly, the D131A CbsA mutant protein displayed the enzymatic activity of a typical endoglucanase. D131A CbsA was as proficient as wild‐type (Wt) CbsA in inducing rice immune responses, but was deficient in virulence‐promoting activity. This indicates that the specific exoglucanase activity of the Wt CbsA protein is required for this protein to promote the growth of Xoo in rice.     

The role of type III effectors from Xanthomonas axonopodis pv. manihotis in virulence and suppression of plant immunity

Xanthomonas axonopodis pv. manihotis (Xam) causes cassava bacterial blight, the most important bacterial disease of cassava. Xam, like other Xanthomonas species, requires type III effectors (T3Es) for maximal virulence. Xam strain CIO151 possesses 17 predicted T3Es belonging to the Xanthomonas outer protein (Xop) class. This work aimed to characterize nine Xop effectors present in Xam CIO151 for their role in virulence and modulation of plant immunity. Our findings demonstrate the importance of XopZ, XopX, XopAO1 and AvrBs2 for full virulence, as well as a redundant function in virulence between XopN and XopQ in susceptible cassava plants. We tested their role in pathogen‐associated molecular pattern (PAMP)‐triggered immunity (PTI) and effector‐triggered immunity (ETI) using heterologous systems. AvrBs2, XopR and XopAO1 are capable of suppressing PTI. ETI suppression activity was only detected for XopE4 and XopAO1. These results demonstrate the overall importance and diversity in functions of major virulence effectors AvrBs2 and XopAO1 in Xam during cassava infection.     

Highly efficient genome editing in Xanthomonas oryzae pv. oryzae through repurposing the endogenous type I-C CRISPR-Cas system

Efficient and modular genome editing technologies that manipulate the genome of bacterial pathogens will facilitate the study of pathogenesis mechanisms. However, such methods are yet to be established for Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of rice bacterial blight. We identified a single type I-C CRISPR-Cas system in the Xoo genome and leveraged this endogenous defence system for high-efficiency genome editing in Xoo. Specifically, we developed plasmid components carrying a mini-CRISPR array, donor DNA, and a phage-derived recombination system to enable the efficient and programmable genome editing of precise deletions, insertions, base substitutions, and gene replacements. Furthermore, the type I-C CRISPR-Cas system of Xoo cleaves target DNA unidirectionally, and this can be harnessed to generate large genomic deletions up to 212 kb efficiently. Therefore, the genome-editing strategy we have developed can serve as an excellent tool for functional genomics of Xoo, and should also be applicable to other CRISPR-harbouring bacterial plant pathogens.     

水稻病程相关PR1家族蛋白质在叶片生长及与白叶枯病菌互作反应中的表达

病程相关(PR)蛋白质经常被用作抗病反应的分子标记。利用免疫印迹(WB)技术检测了7个PR1家族蛋白质在水稻(Oryza sativa)叶片生长及与白叶枯病菌互作反应过程中的表达,发现6个PR1家族蛋白质在叶片生长中有表达。检测PR1蛋白质在Xa21介导的抗白叶枯病过程中的表达,结果显示PR1#052、PR1#072、PR1#073和PR1#121四个蛋白质在抗病反应后期呈上调或诱导表达,PR1#071则表达下调。进一步比较它们在抗病、感病和对照(Mock)反应中的表达丰度,发现在抗病和感病反应中的变化幅度均明显大于对照反应,推测这些PR蛋白质在水稻-白叶枯病菌互作反应中发挥作用。另外,对PR1基因上游启动子区的cis元件进行了分析。该研究初步揭示了水稻PR1家族蛋白质的表达谱,为进一步了解PR1蛋白质的功能提供了线索。     

水稻白叶枯病菌EAL结构域蛋白VieAxoo基因缺失突变和功能分析

摘要：【目的】旨在揭示水稻白叶枯病菌(Xanthomonas oryzae pv. oryzae, 简称Xoo) 环鸟苷二磷酸（c-di-GMP）信号蛋白VieAxoo的生物学功能。【方法】本研究通过标记置换法对vieAxoo基因(PXO_04753)进行了缺失突变研究,采用表性测定进行了部分功能鉴定。【结果】从野生型菌株PXO99A中克隆的vieAxoo基因序列与其它病原黄单胞菌的同源序列高度保守。VieAxoo具有参与c-di-GMP降解的磷酸二酯酶（PDE）EAL结构域和磷酸信号识别受体REC结构域     

Artificial Agrobacterium tumefaciens strains exhibit diverse mechanisms to repress Xanthomonas oryzae pv. oryzae‐induced hypersensitive response and non‐host resistance in Nicotiana benthamiana

Xanthomonas oryzae pv. oryzae (Xoo) rapidly triggers a hypersensitive response (HR) and non‐host resistance in its non‐host plant Nicotiana benthamiana. Here, we report that Agrobacterium tumefaciens strain GV3101 blocks Xoo‐induced HR in N. benthamiana when pre‐infiltrated or co‐infiltrated, but not when post‐infiltrated at 4 h after Xoo inoculation. This suppression by A. tumefaciens is local and highly efficient to Xoo. The HR‐inhibiting efficiency of A. tumefaciens is strain dependent. Strain C58C1 has almost no effect on Xoo‐induced HR, whereas strains GV3101, EHA105 and LBA4404 nearly completely block HR formation. Intriguingly, these three HR‐inhibiting strains employ different strategies to repress HR. Strain GV3101 displays strong antibiotic activity and thus suppresses Xoo growth. Comparison of the genotype and Xoo antibiosis activity of wild‐type A. tumefaciens strain C58 and a set of C58‐derived strains reveals that this Xoo antibiosis activity of A. tumefaciens is negatively, but not solely, regulated by the transferred‐DNA (T‐DNA) of the Ti plasmid pTiC58. Unlike GV3101, strains LBA4404 and EHA105 exhibit no significant antibiotic effect on Xoo, but rather abolish hydrogen peroxide accumulation. In addition, expression assays indicate that strains LBA4404 and EHA105 may inhibit Xoo‐induced HR by suppression of the expression of Xoo type III secretion system (T3SS) effector genes hpa1 and hrpD6. Collectively, our results unveil the multiple levels of effects of A. tumefaciens on Xoo in N. benthamiana and provide insights into the molecular mechanisms underlying the bacterial antibiosis of A. tumefaciens and the non‐host resistance induced by Xoo.