大豆斑疹病菌harpin编码基因的克隆与特性研究

根据黄单胞菌harpin编码基因的同源性,设计简并引物,采用PCR方法从大豆斑疹病菌（Xanthomonas axonopodis pv.glycines, Xag）中克隆了402 bp的[STBX]hpa1[STBZ]同源基因,构建于表达载体pET30(a)上经转化大肠杆菌BL21菌株,获得基因工程菌BHR_3。基因工程菌诱导表达后经收集菌体和破碎细胞,得到表达产物为151kD的蛋白质。该蛋白质富含甘氨酸,不含半胱氨酸,对热稳定,对蛋白酶K敏感,可在非寄主烟草上激发过敏反应。激发的过敏反应需要植物体内水杨酸的积累,还可被真核生物代谢抑制剂抑制。序列比较显示,该基因与Xag中hpaG基因相同,与其它黄单胞菌中的hpa1基因有51.4%～93.8%的同源性,与其它革兰氏阴性植物病原细菌的harpin编码基因无同源性。据此把该基因产物鉴定为harpinXag。黄单胞菌harpin蛋白质序列比较发现,GG_GGG基序的多少并不是harpin蛋白的唯一特性。这为利用harpin蛋白开展植物病害控制的基因药物学设计提供了科学线索。     

Recent advances in the understanding of Xanthomonas citri ssp. citri pathogenesis and citrus canker disease management

Taxonomic status : Bacteria; Phylum Proteobacteria; Class Gammaproteobacteria; Order Xanthomonadales; Family Xanthomonadaceae; Genus Xanthomonas; Species Xanthomonas citri ssp. citri (Xcc). Host range : Compatible hosts vary in their susceptibility to citrus canker (CC), with grapefruit, lime and lemon being the most susceptible, sweet orange being moderately susceptible, and kumquat and calamondin being amongst the least susceptible. Microbiological properties : Xcc is a rod‐shaped (1.5–2.0 × 0.5–0.75 µm), Gram‐negative, aerobic bacterium with a single polar flagellum. The bacterium forms yellow colonies on culture media as a result of the production of xanthomonadin. Distribution : Present in South America, the British Virgin Islands, Africa, the Middle East, India, Asia and the South Pacific islands. Localized incidence in the USA, Argentina, Brazil, Bolivia, Uruguay, Senegal, Mali, Burkina Faso, Tanzania, Iran, Saudi Arabia, Yemen and Bangladesh. Widespread throughout Paraguay, Comoros, China, Japan, Malaysia and Vietnam. Eradicated from South Africa, Australia and New Zealand. Absent from Europe.     

Distribution and Races of Xanthomonas axonopodis pv. malvacearum on Cotton (Gossypium hirsutum) in Uganda

Surveys in 1995 and 1996 showed that bacterial blight caused by Xanthomonas axonopodis pv. malvacearum occurs throughout the main cotton growing areas of Uganda, causing seedling blight, angular leaf spot and bacterial boll rot. During the vegetative and early fruiting stages of crop growth, severe symptoms of `blackarm' spread from leaves to the stem, causing loss of fruiting branches. A set of Upland cotton cultivars ( Gossypium hirsutum ) were then used to determine the races of the blight bacterium present in Uganda. Many of the isolates induced moderate to severe symptoms on all the test hosts except 101–102B, indicating infection with race 10 or 18. The next most common isolate was race 7. Races 16 and 6 were also identified and 23% of isolates caused symptoms on all the differential cultivars including 101–102B, results indicating the presence of a race of the pathogen which may be the same as that identified in countries neighbouring Uganda and designated as race 20.     

Proteins induced by Xanthomonas axonopodis pv. passiflorae with leaf extract of the host plant (Passiflorae edulis)

Two-dimensional gel electrophoresis was used to identify differentially displayed proteins during treatment of Xanthomonas axonopodis pv. passiflorae in media containing leaf extract of the compatible (passion fruit) and incompatible (tomato) hosts. The results showed that at different times of treatment (5, 25 and 45 h) the global expression of proteins was almost identical in cells grown in minimal medium (MM) and in medium containing leaf extract of the incompatible host (MMT). The protein patterns of cells grown in medium containing passiflorae (MMP) leaf extract and MM were also compared enabling the detection of 17 differential spots. Most of the proteins were induced at earlier times of incubation (5 h) and maintained until 45 h in MMP. By using another carrier ampholyte range, seven additional proteins were identified in MMP treated cells. Five proteins, including one constitutive, two induced and two up-regulated in MMP were microsequenced. All sequences were found in the genome of xanthomonads sharing high level of identity (88-100%). Fructose biphosphate aldolase was expressed in all media employed. A putative membrane-related protein and a hypothetical protein were novel proteins induced specifically by the passiflorae extract. An inorganic pyrophosphatase and a hypothetical protein that showed similarity to the yciF gene of Salmonella thyphimurium were up-regulated in MMP.     

Bacterial and plant natriuretic peptides improve plant defence responses against pathogens

Plant natriuretic peptides (PNPs) have been implicated in the regulation of ions and water homeostasis, and their participation in the plant immune response has also been proposed. Xanthomonas citri ssp. citri contains a gene encoding a PNP‐like protein (XacPNP) which has no homologues in other bacteria. XacPNP mimics its Arabidopsis thaliana homologue AtPNP‐A by modifying host responses to create favourable conditions for pathogen survival. However, the ability of XacPNP to induce plant defence responses has not been investigated. In order to study further the role of XacPNP in vivo, A. thaliana lines over‐expressing XacPNP, lines over‐expressing AtPNP‐A and AtPNP‐A‐deficient plants were generated. Plants over‐expressing XacPNP or AtPNP‐A showed larger stomatal aperture and were more resistant to saline or oxidative stress than were PNP‐deficient lines. In order to study further the role of PNP in biotic stress responses, A. thaliana leaves were infiltrated with pure recombinant XacPNP, and showed enhanced expression of genes related to the defence response and a higher resistance to pathogen infections. Moreover, AtPNP‐A expression increased in A. thaliana on Pseudomonas syringae pv. tomato (Pst) infection. This evidence led us to analyse the responses of the transgenic plants to pathogens. Plants over‐expressing XacPNP or AtPNP‐A were more resistant to Pst infection than control plants, whereas PNP‐deficient plants were more susceptible and showed a stronger hypersensitive response when challenged with non‐host bacteria. Therefore, XacPNP, acquired by horizontal gene transfer, is able to mimic PNP functions, even with an increase in plant defence responses.     

柑桔溃疡病菌滚环扩增检测体系的建立

根据柑桔溃疡病菌(Xanthomonas axonopodis pv.citri,Xac)独有的蛋白基因序列和锁式探针公共连接序列分别设计特异性的锁式探针及其扩增引物,优化系列反应条件,建立了特异性的柑桔溃疡病菌滚环扩增体系。初步检测结果表明该体系能够特异性地检出Xac的菌体细胞及其DNA,而检测不出供试的其它植物病原细菌和柑桔叶面常见的多种附生细菌;对Xac靶片段克隆质粒DNA的检测灵敏度为102copy/μL,对Xac菌悬液的检测灵敏度为20cfu/μL,比常规PCR的检测灵敏度稍高。用滚环扩增技术和常规PCR技术对田间采集的实际样品进行了检测,两种方法的检测结果没有显著差异(P>0.01)。由于锁式探针的公共连接序列对扩增的条件要求一致,本体系的建立可以为植物病原微生物多靶标检测和病害检疫检验提供新的技术支撑。     

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.