Establishment of a system for screening and identification of novel bactericide targets in the plant pathogenic bacterium Xanthomonas oryzae pv. oryzae using Tn-seq and SPR

Xanthomonas spp. cause severe bacterial diseases. However, effective strategies for prevention and management of these diseases are scarce. Thus, it is necessary to improve the efficiency of control of diseases caused by Xanthomonas. In this study, Xanthomonas oryzae pv. oryzae(Xoo), which causes rice bacterial leaf blight, has been studied as a representative. A transposon insertion library of Xoo, comprising approximately 200,000 individual insertion mutants, was generated. Transposon sequencing data indicated that the mariner C9 transposase mapped at 35.7–36.4% of all potential insertion sites, revealing 491 essential genes required for the growth of Xoo in rich media. The results show that, compared to the functions of essential genes of other bacteria, the functions of some essential genes of Xoo are unknown, 25 genes might be dangerous for the Xanthomonas group, and 3 are specific to Xanthomonas. High-priority candidates for developing broad-spectrum, Xanthomonas-specific, and environment-friendly bactericides were identified in this study. In addition, this study revealed the possible targets of dioctyldiethylenetriamine using surface plasmon resonance(SPR) in combination with high performance liquid chromatography–mass spectrometry(HPLC–MS). The study also provided references for the research of some certain bactericides with unknown anti-bacterial mode of action. In conclusion, this study urged a better understanding of Xanthomonas,provided meaningful data for the management of bacterial leaf blight, and disclosed selected targets of a novel bactericide.

Molecular Screening of Rice Cultivated in Benin for the Identification of Xanthomonas oryzae Pv. oryzae and Bacterial Leaf Blight Resistance Genes

One of the most devastating diseases of rice worldwide is bacterial blight (BLB) caused by Xanthomonas oryzae pv. Oryzae (Xoo). In Benin, Xoo was first described in 2013 on wild rice Oryzae longistaminata. So far, no study has been done on Beninese Xoo strains. We do not know whether the pathogen has already passed into the rice varieties grown, or if they are exposed to other bacteria. Whereas the use of resistant varieties, carrying resistance genes, is the only highly effective and environmentally friendly way to control this disease, no information is available on these Xoo resistance genes in rice varieties grown in Benin apart from the one we recently. This study aims to identify Beninese Xoo strains, causing BLB and screen rice varieties grown in Benin for the main resistance genes. Diseased rice leaves showing typical symptoms of fire blight collected from different rice fields in the three phytogeographic areas of Benin were analyzed by PCR for Xoo-specific sequence identification. Furthermore, seventy-five collected rice accessions were screened to identify xa5, Xa7, xa13, and Xa21 resistance genes to Xoo. The results reveal that Xanthomonas oryzae was identified in two fields in Banikouara and one in Malanville. On the other hand, Sphingomonas sp. has been identified in several other rice fields in Benin. Forty-seven of seventy-five rice accessions examined (62.66%) carried Xoo resistance genes with 3 (4%) and 40 (53.33%) of xa5 and Xa21 respectively. None of the accessions had either Xa7 or xa13 resistance genes. Three accessions possess both xa5 and Xa21 genes. Isogenic lines IRBB60 and IRBB21, supposed to be a positive control, presented a Xoo sensitivity allele. These results indicate that Xoo has moved from the wild rice variety to the cultivated variety in northern Benin and varietal improvement programs must be implemented with varieties having several resistance genes for the efficient response against a possible BLB pandemic in Benin.

黄单胞菌Xanthomonas campestris pv.raphani 756C中Ⅵ型分泌蛋白的生物信息学分析

为明确黄单胞菌Xanthomonas campestris pv.raphani 756C(Xcr)中存在的Ⅵ型分泌蛋白(Tss)数量及其所具有的信号肽、保守motif等信息以及该菌中Tss与其他病菌中同源序列之间的遗传关系,利用关键词对Xcr蛋白质数据库进行搜索,并对Xcr中Tss氨基酸序列开展信号肽、跨膜结构域以及保守基序(motif)的生物信息学分析,同时,对Xcr中所含有的Tss与其他病原菌中同源序列之间的遗传关系进行分析。明确Xcr中存在3个Tss,分别命名为TssA、TssB、TssC,上述Tss均含有高于50%比例的!螺旋结构,均定位在细胞膜上以及具有3个保守motif,而就信号肽而言,仅TssC含有明显的信号肽序列。Xcr中的Tss与Xcc、Xca等黄单胞菌属病菌中的Tss具有较近的亲缘关系。

冰核活性细菌Xanthomonas ampelinaTS206实验室水平发酵生产工艺条件优化的研究

优化冰核活性细菌XanthomonasampelinaTS206实验室水平发酵生产的工艺条件为将冰核活性细菌应用在工业化生产提供了重要的技术资料。本文对冰核活性细菌XanthomonasampelinaTS206实验室水平的发酵生产工艺条件进行了系统的研究,结果表明这种冰核活性细菌的最适培养条件为:培养温度18℃,装液量40ml/250ml,摇床转速180r/min,接种量3%,发酵液的初始pH8。实验还确定了冰核活性细菌在发酵48h时达到对数生长期。采用上述发酵条件培养XanthomonasampelinaTS206可以使细菌在冰核活性不降低的条件下发酵液的菌体浓度达到2.87×1010个/ml,菌体干重增加了37.7%。

十字花科蔬菜黑腐病菌(Xanthomonas campestris pv. campestris)AFLP分析体系的建立及优化

以英国华威大学收集的十字花科蔬菜黑腐病菌野油菜黄单胞菌野油菜致病变种(Xanthomonas campestris pv.campestris)的6个生理小种菌株为材料,优化了该类菌种的AFLP分析体系包括DNA的提取、MseI/EcoRI酶切时间、扩增反应体系的组成及染色方法等步骤,建立了一套适于该菌种的AFLP分析体系。该体系中各最优因素为:酶切体系为50μL,模板DNA用量为600ng,酶切体系中MseI和EcoRI各加入5U,酶切温度为37℃,反应时间为4～6h;预扩增产物稀释10倍进行选择性扩增;检测方法为银染法,银染液中硝酸银含量为8g/L且染色时间是15min时效果最佳,该体系的建立为该类菌种的分子水平遗传多样性研究提供了技术支撑。

海藻酸钙固定化对Xanthomonas campestris206冰核活性的影响

应用正交法研究了海藻酸钙固定化对Xanthomonascampestris 2 0 6冰核活性的影响。结果表明 ,对固定化小球冰核活性影响程度大小的顺序依次为 :菌悬液用量 >海藻酸钠用量 >CaCl2 浓度 >固化时间 ,而对渗漏量影响程度大小的顺序依次为 :菌悬液用量 >固化时间 >CaCl2 浓度 >海藻酸钠用量。研究中还发现 ,将冰核活性细菌固定化在热稳定性方面实际意义不大。

EPPO新增A1类检疫性有害生物—Xanthomonas axonopodis pv. allii

Xanthomonas axonopodis pv. allii主要引起洋葱细菌性叶枯病,是EPPO新增加的A1类有害生物。本文对该菌的分布、寄主、症状、病原、发生规律、传播途径、检测鉴定、防治方法等方面进行了综述。

黑腐病菌(Xanthomonas campestris)与拟南芥(Arabidopsis thaliana)品种间的致病性测定

甘蓝黑腐病菌(Xanthomonas campeatris pv.campestris)主要以十字花科植物为寄主.引起黑腐病的病原菌,是在世界范围对农业造成严重危害的病原菌之一.拟南芥(Arabidopsis thaliana)是十字花科植物,做为经典遗传学实验材料已有40多年历史.它具有较少染色体组和重复 DNA 序列,易诱变、易种植、个体小、

A Non-Marker Mutagenesis Strategy to Generate Poly-hrp Gene Mutants in the Rice Pathogen Xanthomonas oryzae pv. oryzicola

Xanthomonas oryzae pv.oryzicola （Xoc）,the critical pathogen causing bacterial leaf streak in rice,possesses a hrp cluster that is responsible for triggering hypersensitive response （HR） in non-host tobacco and pathogenicity in host rice,and is considered to be one of the model pathogens in the rice model plant.Here,we developed a high-throughput mutagenesis system using a two-step integration mediated by a novel suicide vector pKMS1.It was used to generate single or poly-gene mutants of hpa1,hpa2,hrcV,hrpE,hpaB,and hrpF gene for functional analysis.In total,five single,four double,and two triple hrp gene mutants were constructed.The double and triple hrp gene deletion mutants triggered novel phenotypes in planta.Our data suggest that pKMS1 is a useful tool for non-marker mutagenesis of multiple genes in Xoc.

Action modes of transcription activator-like effectors(TALEs) of Xanthomonas in plants

Plant-pathogenic Xanthomonas infects a wide variety of host plants and causes many devastating diseases on crops. Transcription activator-like effectors（TALEs） are delivered by a type III secretion system（T3 SS） of Xanthomonas into plant nuclei to directly bind specific DNA sequences（TAL effector-binding elements, EBEs） on either strand of host target genes with an unique modular DNA-binding domain and to bidirectionally drive host gene transcription. The target genes in plants consist of host susceptibility（S） genes promoting disease（ETS） and resistance（R） genes triggering defense（ETI）. Here we generally summarized the discovery of TALEs in Xanthomonas species, their functions in bacterial pathogenicity in plants and their target genes in different host plants, and then focused on the newly revealed modes of protein action in triggering or suppressing plant defense.

HrcQ is necessary for Xanthomonas oryzae pv. oryzae HR-induction in non-host tobacco and pathogenicity in host rice

Bacterial blight, caused by Xanthomonas oryzae pv. oryzae(Xoo), is one of the most destructive diseases of rice(Oryza sativa L.) worldwide. The type III secretion system(T3SS) of Xoo, encoded by the hrp(hypersensitive response and pathogenicity) genes, plays critical roles in conferring pathogenicity in host rice and triggering a hypersensitive response(HR) in non-host plants. To investigate the major genes conferring the pathogenicity and avirulence of Xoo, we previously constructed a random Tn5-insertion mutant library of Xoo strain PXO99A. We report here the isolation and characterization of a Tn5-insertion mutant PXM69. Tn5-insertion mutants were screened on indica rice JG30, which is highly susceptible to PXO99A, by leaf-cutting inoculation.Four mutants with reduced virulence were obtained after two rounds of screening. Among them, the mutant PXM69 had completely lost virulence to the rice host and ability to elicit HR in non-host tobacco. Southern blotting analysis showed a single copy of a Tn5-insertion in the genome of PXM69. PCR walking and sequencing analysis revealed that the Tn5 transposon was inserted at nucleotide position 70,192–70,201 in the genome of PXO99A, disrupting the type III hrc(hrp-conserved) gene hrcQ, the first gene in the D operon of the hrp cluster in Xoo. To confirm the relationship between the Tn5-insertion and the avirulence phenotype of PXM69, we used the marker exchange mutagenesis to create a PXO99Amutant, ΔhrcQ::KAN, in which the hrcQ was disrupted by a kanamycin-encoding gene cassette at the same site as that of the Tn5-insertion. ΔhrcQ::KAN showed the same phenotype as mutant PXM69. Reintroduction of the wild-type hrcQ gene partially complemented the pathogenic function of PXM69. RT-PCR and cellulase secretion assays showed that the Tn5-disruption of hrcQ did not affect transcription of downstream genes in the D operon and function of the type II secretion system. Our results provide new insights into the pathogenic functions of clustered hrp genes in Xoo.

An Inner Membrane Protein(Imp) of Xanthomonas oryzae pv. oryzicola Functions in Carbon Acquisition, EPS Production, Bacterial Motility and Virulence in Rice

Xanthomonas oryzae pv. oryzicola （Xoc） causes bacterial leaf streak, a devastating disease in rice-growing regions worldwide. A Tn5-insertion mutant in Xoc_3248, encoding an inner membrane protein （Imp）, showed reduced virulence in rice. To explore the potential function of this gene in virulence, a deletion mutant R？imp was constructed in the wild-type RS105. The R？imp mutant was signiifcantly impaired for bacterial virulence and growth in planta. The mutation in imp made the pathogen insufifciently utilize glucose, fructose, mannose or pyruvate as a sole carbon source, leading to less extracellular polysaccharide （EPS） production and reduced motility. The deifciencies noted for the mutant were restored to wild-type levels when imp was introduced in trans. Transcription of imp was signiifcantly declined when hrpG and hrpX was mutated and the expression of hrpG and hrpX was also signiifcantly declined when imp was deleted. Cell sublocalization in planta showed Imp membrane-binding feature. These results suggest that Imp is a virulence factor with roles in the catabolism of sugars, EPS production, and bacterial motility.

Molecular detection of Xanthomonas oryzae pv.oryzae, Xanthomonas oryzae pv. oryzicola, and Burkholderia glumae in infected rice seeds and leaves

The polymerase chain reaction(PCR) is particularly useful for plant pathogen detection. In the present study, multiplex PCR and SYBR Green real-time PCR were developed to facilitate the simultaneous detection of three important rice pathogens, Xanthomonas oryzae pv.oryzae, X. oryzae pv. oryzicola, and Burkholderia glumae. The unique PCR primer sets were designed from portions of a putative glycosyltransferase gene of X. oryzae pv. oryzae, an Avr Rxo gene of X. oryzae pv. oryzicola, and an internal transcribed spacer(ITS) sequence of B. glumae. Using a multiplex PCR assay, X. oryzae pv. oryzae, X. oryzae pv. oryzicola, and B. glumae were detected in one PCR reaction that contained the newly developed primer set mix. Using SYBR Green real-time PCR assays, X. oryzae pv. oryzae, X. oryzae pv. oryzicola, and B. glumae were detected at 1, 1, and 10 fg μL-1, respectively. These newly designed molecular assays are sensitive and could be reliable tools for pathogen detection and disease forecasting.

An improved protein expression system for T3SS genes regulation analysis in Xanthomonas oryzae pv. oryzae

Xanthomonas oryzea pv.oryzae(Xoo)is the causal agent of bacterial blight of rice,which is a significant threat to many of rice-growing regions.The type Ⅲ secretion system(T3SS)is an essential virulence factor in Xoo.Expression of the T3SS is often induced in the host environment or in hrp-inducing medium but is repressed in nutrient-rich medium.The elucidation of molecular mechanism underlying induction of T3SS genes expression is a very important step to lift the veil on global virulence regulation network in Xoo.Thus,an efficient and reliable genetic tool system is required for detection of the T3SS proteins.In this study,we constructed a protein expression vector pH3-flag based on the backbone of pHM1,a most widely used vector in Xoo strains,especially a model strain PXO99A.This vector contains a synthesized MCS-FLAG cassette that consists of a multiple cloning site(MCS),containing a modified pUC18 polylinker,and Flag as a C-terminal tag.The cassette is flanked by transcriptional terminators to eliminate interference of external transcription enabling detection of accurate protein expression.We evaluated the potential of this expression vector as T3SS proteins detection system and demonstrated it is applicable in the study of T3SS genes expression regulation in Xoo.This improved expression system could be very effectively used as a molecular tool in understanding some virulence genes expression and regulation in Xoo and other Xanthomonas spp.

Citron C-05 inhibits both the penetration and colonization of Xanthomonas citri subsp.citri to achieve resistance to citrus canker disease

Citrus canker,caused by Xanthomonas citri subsp.citri(Xcc),is a serious bacterial disease that affects citrus production worldwide.Citron C-05(Citrus medica)is the only germplasm in the Citrus genus that has been identified to exhibit strong resistance to Xcc.However,it has not been determined when,where,and how Xcc is restricted in the tissues of Citron C-05 during the infection process.In the present study,we investigated the spatiotemporal growth dynamics of an eGFP-labeled virulent Xcc(eGFP-Xcc)strain in Citron C-05 along with five susceptible biotypes(i.e.,lemon,pummelo,sour orange,sweet orange,and ponkan mandarin)upon inoculation via the spraying or leaf infiltration of a bacterial suspension.The results from extensive confocal laser scanning microscopy analyses showed that while Xcc grew rapidly in plants of all five susceptible genotypes,Xcc was severely restricted in the epidermal and mesophyll cell layers of the leaves of Citron C-05 in the early stage of infection.Not surprisingly,resistance against Xcc in Citron C-05 was found to be associated with the production of reactive oxygen species and hypersensitive response-like cell death,as well as greater upregulation of several defense-related genes,including a pathogenesis-related gene(PR1)and a glutathione S-transferase gene(GST1),compared with sweet orange as a susceptible control.Taken together,our results not only provide further valuable details of the spatiotemporal dynamics of the host entry,propagation,and spread of Xcc in both resistant and susceptible citrus plants but also suggest that resistance to Xcc in Citron C-05 may be attributed to the activation of multiple defense mechanisms.

Comparison of Positions of QTLs Conferring Resistance to <i>Xanthomonas campestris</i>pv. <i>campestris</i>in <i>Brassica oleracea</i>

Black rot, caused by Xanthomonas campestris pv. campestris (Xcc) is possibly the most important disease of Brassica worldwide. To compare chromosomal positions of Xcc resistance loci in Brassica oleracea between the present and published studies and to develop marker assisted selection (MAS) to resistance against Xcc race 1, we constructed a B. oleracea map, including pW, pX and BoCL markers that were closely linked to previously reported Xcc resistance QTLs. We also analyzed Xcc resistance QTLs by improving our previously reported map derived from the cross of a susceptible double-haploid line (GC P09) with a resistant double-haploid line (Reiho P01). In the nine linkage groups obtained (C1-C9), the major QTL, XccBo(Reiho)2, was derived from Reiho with a maximum LOD score (7.7) in C8. The QTL (LOD 4.4) located in C9, XccBo(GC)1 was derived from the susceptible GC. The other QTL (LOD 4.4), XccBo(Reiho)1, was found in C5. Based on common markers, it was possible to compare our finding Xcc resistance QTLs with the B. oleraceaXcc loci reported by previous authors;XccBo(Reiho)1 and XccBo(GC)1 may be identical to the Xcc resistance QTLs reported previously or a different member contained in the same resistance gene cluster. Our map includes public SSR markers linked to Xcc resistance genes that will promote pyramiding Xcc resistance genes in B. oleracea. The present study will also contribute to a better understanding of genetic control of Xcc resistance.

Xoryp_08180 of Xanthomonas oryzae pv.oryzicola,Encoding a Hypothetical Protein,is Regulated by HrpG and HrpX and Required for Full Virulence in Rice

Xanthomonas oryzae pv.oryzicola（Xoc） causes a destructive bacterial leaf streak disease in rice.Some of the gene products annotated as hypothetical proteins in the genome of Xoc may contribute to its virulence in rice.A mutant,Mxoc1679,screened from our previous Tn5-tagged mutant library for Xoc strain RS105,showed reduced virulence in rice.In this mutant,a gene named as Xoryp_08180 was disrupted by Tn5 insertion.Xoryp_08180 encodes a 1 306-aa hypothetical protein which is highly conserved in Xanthomonas spp.Non-polar mutation of Xoryp_08180 in RS105 strain led to a significant reduction in bacterial virulence and growth in rice,a delayed hypersensitive response（HR） in non-host tobacco,and a decrease in extracellular protease activity.The deficiencies above were restored to wild-type level in the complementary strain by expressing Xoryp_08180 in trans.In addition,the expression of Xoryp_08180 was repressed in hrpG and hrpX mutants in planta but not in a nutrient-rich condition.These results suggested that Xoryp_08180 is a virulence factor required for extracellular protease production,HR induction and full virulence of Xoc.

The Twin-Arginine Translocation (Tat) Pathway Is Essential for Virulence,Flagellation,and Chemotaxis in Xanthomonas oryzae pv. oryzicola Strain RsGD42

Bacterial leaf streak, caused by Xanthomonas oryzae pv. oryzicola, is an important disease of rice （Oryza sativa）. Genetic determinants （tatABC genes） of the twin-arginine translocation （Tat） pathway from X. oryzae pv. oryzicola strain RsGD42 were cloned and characterized, meanwhile, a tatC disruption mutant was generated. The tatC mutant lacked detectable flagella and was highly impaired in motility and chemotaxis. Furthermore, it was observed that the tatC mutant exhibited a reduced production of extracellular polysaccharide （EPS） and a significant reduction of virulence on adult rice plants compared to wild type strain. However, the tatC mutation in X. oryzae pv. oryzieola strain RsGD42 did not affect the growth rate and the ability to induce hypersensitive response （HR） in nonhost tobacco （Nicotiana tabacum L. cv. Samsun）. In conclusion, the data indicated that the Tat pathway significantly contributed to the virulence of X. oryzae pv. oryzicola.

Exchangeability of Two hrp Gene Fragments from Xanthomonas oryzae pv.oryzae and pv.oryzicola for Hypersensitive Response on Tobacco and Pathogenicity on Rice

hrp mutants were produced from strain JXOIII of Xanthomonas oryzae pv. oryzae (Xoo) and strain RS105 of X.o. pv. oryzicola (Xooc), respectively, by using diethyl sulfate (DES) as a mutagenic che mical. All the hrp mutants lost their pathogenicity on a susceptible host plant, rice (Shanyou63), and elicitation of the hypersensitive response (HR) on a nonhost plant, tobacco (NC89). Extracellular enzyme (amy lase, pectate lyase, proteinase, cellulase and lipase) activities of all the hrp mutants were similar to those of the corresponding wild type strains. The response of tobacco to cell sonicated integrations of the wild type strains and the hrp mutants demonstrated that there existed an HR eliciting substance which was heat stable and sensitive to protease. No HR appeared on tobacco after infiltration of the lipopolysaccharide (LPS) of both the wild strains and hrp mutants into tobacco leaves. The ability of the Xooc hrp mutants to induce HR on tobacco and cause streak disease on rice was restored by complementation with pUHRX245 from JXOIII genomic DNA library and by pUHRS138 from RS105 genomic DNA library, respectively. Subcloning of a 38.6 kb hrp fragment insert in pUHRX245 and a 39.3 kb insert in pUHRS138 revealed that a 3.3 kb Sac Ⅰ fragment from pUHRX245 and a 4.5 kb Bam HⅠ Kpn Ⅰ fragment from pUHRS138 were the minimal functional portions required for restoration of the ability of Xooc hrp mutants to induce HR on tobacco and cause disease on rice. The disease symptom caused by the conjugant (M1005 plus 3.3 kb) on rice was similar to that caused by the wild type of Xooc. It suggests that the two fragments contain the same hrp gene(s) and are responsible reciprocally for HR induction on tobacco and pathogenicity on rice.

Virulence of Xanthomonas oryzae pv. oryzae on Rice Near-lsogenic Lines with Single Resistance Gene and＇Pyramiding Lines in China

Ninety one isolates of Xanthomonas oryzae pv. oryzae were collected from different rice- growing regions in China and determined for their virulence on 24 rice near-isogenic lines containing single resistance gene and 2-4 genes: IRBB1 (Xa1), IRBB2 (Xa2), IRBB3 (Xa3), IRBB4 (Xa4), IRBB5 (xa5), IRBB7 (Xa7), IRBB8 (xa8), IRBB10 (Xa10), IRBB11 (Xa11), IRBB13 (xa13), IRBB14 (Xa14), IRBB21 (Xa21), IR24 (Xa18), IRBB50 (Xa4 + xa5), IRBB51 (Xa4 + xa13), IRBB52 (Xa4 + Xa21), IRBB53 (xa5 + xa13), IRBB54 (xa5 + Xa21), IRBB55 (xa13 + Xa21), IRBB56 (Xa4 + xa5 + xa13), IRBB57 (Xa4 + xa5 + Xa21), IRBB58 (Xa4 + xa13 + Xa21), IRBB59 (xa5 + xa13 + Xa21) and IRBB60 (Xa4 + xa5 + xa13 + Xa21). The results showed that most isolates were less virulent on lines with more than one genes pyramided than those with single resistance gene. The isolates tested were more virulent on IR24 and IRBB10, less virulent on IRBB5, IRBB7 and IRBB21. Based on interactions between isolates and rice near-isogenic lines, 7 cultivars with single gene (IRBB5, IRBB4, IRBB3, IRBB14, IRBB2, IRBB1 and IR24) were chosen as the differentials, and the tested isolates were classified into 7 virulence groups. The reaction patterns of the 7 groups in order were: RRRRRRR, RRRRRRS, RRRRRSS, RR/SRRSSS, RRRSSSS, RRSSSSS, RSSSSSS. The virulence frequencies were 7.69, 6.59, 14.29, 12.09, 14.29, 28.57 and 16.48% respectively. The elementary system for races identification has been established in China based on the results. It will be possible to compare with races in other countries, and the results will facilitate the development of rice resistance breeding to bacterial blight in China.