The small and large subunits of carbamoyl-phosphate synthase exhibit diverse contributions to pathogenicity in Xanthomonas citri subsp.citri

Carbamoyl-phosphate synthase plays a vital role in the carbon and nitrogen metabolism cycles. In Xanthomonas citrisubsp. citri, carA and carB encode the small and large subunits of carbamoyl-phosphate synthase, respectively. The deletion mutation of the coding regions revealed that carA did not affect any of the phenotypes, while carB played multiple roles in pathogenicity. The deletion of carB rendered the loss of pathogenicity in host plants and the ability to induce a hyper- sensitive reaction in the non-hosts. Quantitative reverse transcription-PCR assays indicated that 11 hrp genes coding the type Ill secretion system were suppressed when interacting with citrus plants. The mutation in carB also affected bacterial utilization of several carbon and nitrogen resources in minimal medium MMX and extracellular enzyme activities. These data demonstrated that only the large subunit of carbamoyl-phosphate synthase was essential for canker development by X. citri subsp, citri.

Characterization of Xanthomonas citri pv.citri from China based on spoligotyping

Xanthomonas citri pv.citri(Xcc),a gram-negative bacterium,is the causal agent of citrus canker,one of the most devastating diseases threatening the citrus industry worldwide.Understanding the diversity and population structure of Xcc is a prerequisite for disease epidemiological monitoring and effective disease management.Recent characterization of the clustered regularly interspaced short palindromic repeats(CRISPR)/cas(CRISPR-associated proteins genes)system with a highly variable repeat number among species provides a new molecular typing method for bacterial genetic analysis.In this study,we performed systematic in silico analyses of 28 Xcc genomes and identified a credible CRISPR/cas in Xcc strains.Further analysis of CRISPR polymorphisms(repeat number and spacer types)in 129 Xcc A strains collected from six provinces in China identified 15 types of CRISPR arrays with 25 spacers.Phylogenetic analysis of Xcc strains based on the CRISPR locus produced a more reliable and accurate typing result compared to the commonly used loci.In addition,seven associated cas genes—cas1,cas2,cas3,cas4,cas5,cas7(csd2),and cas8(csd1)—were found located adjacent to the CRISPR array.BLAST results showed>99%similarity of seven cas genes among Xcc strains.Homology analysis of spacer sequences showed that six spacers had possible phage/prophage origin.The characterization of the CRISPR/cas system among Xcc strains provided an updated strain typing method for Xcc diversity analysis and yielded a panoramic view of CRISPR evolution for further studies of Xcc-phage interactions.

The Viable but Non-culturable State in Xanthomonas citri subsp, citri is a Reversible State Induced by Low Nutrient Availability and Copper Stress Conditions

Xcc （Xanthomonas citri subsp, citri） causes citrus bacterial canker, a leaf, stem and fruit spotting disease that affects most commercial citrus species and cultivars. Copper compounds, widely used for management of this pathogen, have been reported as inducers of a VBNC （viable but non-culturable state） in plant pathogenic bacteria. VBNC may be considered as a state preceding bacterial death or as a survival mechanism under adverse conditions. Several experiments were performed to characterize the reversibility and persistence of the VBNC state in Xcc. VBNC was induced in low nutrient medium or with amendment of copper at concentrations used for field disease control. The VBNC condition was demonstrated to persist up to 150 days after copper treatment and was reversed after the addition of culture media without copper or amendment with citrus leaf extract. Xcc viability was evaluated by recovery of colonies on culture media, confirmed by membrane integrity, respiratory activity and by real-time RT-PCR targeting a sequence from the gumD gene. Besides, the colonies recovered were pathogenic on citrus leaves. These results confirm that the VBNC state in Xcc is inducible and reversible and therefore may occur in the phyllosphere when Xcc is under copper stress or starvation.

The sigma 54 genes rpoN1 and rpoN2 of Xanthomonas citri subsp. citri play different roles in virulence, nutrient utilization and cell motility

The sigma factor 54（σ^（54）） controls the expression of many genes in response to nutritional and environmental conditions. There are two σ^（54） genes, rpo N1（XAC1969） and rpo N2（XAC2972）, in Xanthomonas citri subsp. citri. To investigate their functions, the deletion mutants ΔrpoN1, ΔrpoN2 and ΔrpoN1N2 were constructed in this study. All the mutants delayed canker development in low concentration inoculation in citrus plants. The bacterial growth of mutants was retarded in the medium supplemented with nitrogen and carbon resources. Under either condition, the influence degree caused by deletion of rpoN 2 was larger than the deletion of rpoN 1. Remarkably, the mutant ΔrpoN 1 showed a reduction in cell motility, while the mutant Δrpo N2 increased cell motility. Our data suggested that the rpoN 1 and rpoN 2 play diverse roles in X. citri subsp. citri.

Comparison of PCR, DIA and Pathogenicity Assay for Detection of Xanthomonas axonopodis pv.citri,the Causal Agent of Citrus Bacterial Canker Disease

Polymerase chain reaction (PCR) approach based on newly designed primers, JYF5/JYR5, wasapplied for specific detection of Xanthomonas axonopodis pv.citri(Xac). The efficiencyand reliability of PCR method were compared with dot immunobinding assay (DIA) andclassical pathogenicity test techniques for detecting suspensions of pure cells of Xacand soaking sap of citrus tissues. Detection sensitivity of PCR was about 4.5 cells or1.56 pg target DNA per reaction which was higher than that of DIA (ca. 450 cells per dot).These three techniques (PCR assay, DIA and Pathogenecity test) could always detect Xacfrom symptomatic citrus samples. Different performances were obtained from citrusmaterials without symptoms, and the positive detection frequency was PCR, DIA andpathogenicity test.

DR5 is a Suitable System for Studying the Auxin Response in the Poncirus trifoliata-Xanthomonas axonopodis pv.citri Interaction

The local auxin distribution characteristics in the roots,stems,and leaves of stably transformed plantlets of trifoliate orange(Poncirus trifoliata)with auxin reporter system DR5::GUS-YFP were elucidated in this research.The auxin response maxima could be observed in the apex of the root tip,primary phloem of the tender stem,and the margin of the young leaves according to the activity of theβ-glucuronidase(GUS)reporter gene triggered by the auxin responsive DR5 promoter.Auxin responses in the apex of the root tips increased when treated with synthetic auxin 1-naphthylacetic acid(NAA),but decreased when treated with the auxin polar transportation inhibitor 2,3,5-triiodobenzoic acid(TIBA).These results indicated that the DR5 reporter system worked in P.trifoliata for auxin distribution and response observation.Trifoliate orange is highly susceptible to citrus canker disease.Auxin accumulation was observed visually in the invasion sites of the detached leaves inoculated with Xanthomonas axonopodis pv.citri(Xac)by GUS staining;the upregulated expression of the YFP,GH3.1,GH3.9,and SAUR genes assessed by quantitative real-time PCR(qRT-PCR)also identified auxin accumulation in the inoculated tissues following Xac infection.Overall,these findings indicated that the plantlets of P.trifoliata engineered with the auxin reporter gene provided a promising system for studying auxin responses during Xac infection.

绿色荧光蛋白(GFP)标记杧果细菌性角斑病病原菌及其定殖规律

为研究杧果细菌性角斑病病菌在杧果不同器官组织中的定殖规律。利用电击转化法将质粒pBBR1MCS2-Tac-EGFP导入野油菜黄单胞菌杧果致病变种(Xanthomonas citri pv.mangiferaeindicae)菌株Xcm003中,通过转化子生长表型和外源基因的PCR鉴定,成功获得带绿色荧光蛋白(GFP)标记的转化子Xcm003-EGFP,采用室内刺伤和盆栽苗灌根接种试验,结合组织学观察法,追踪该病原菌在不同杧果器官组织中的定殖规律。结果表明,病原菌通过伤口侵入杧果果实果皮外层细胞层,随着侵染时间推移,向内层果皮细胞层垂直扩散并定殖,后期病原菌在伤口处大量聚集,出现隆起开裂状病斑。在杧果叶片中,病原菌从伤口侵入叶片上表皮细胞层,随后迁移到叶肉细胞间隙,侵染后期,病原菌在气孔和伤口定殖,造成气孔和伤口堵塞,导致接种点出现黑褐色水浸病斑。盆栽苗灌根后发现,该病原菌可在土壤中定殖,入侵杧果根部,但并不扩散至其他杧果组织,不会为害整株幼苗。说明杧果细菌性角斑病病菌可在不同杧果组织中定殖,但仅表现为局部侵染特性。

Systematic analysis and functional verification of citrus glutathione S-transferases reveals that CsGSTF1 and CsGSTU18contribute negatively to citrus bacterial canker

Citrus bacterial canker(CBC) is resulted from Xanthomonas citri subsp. citri(Xcc) infection and poses a significant threat to citrus production.Glutathione S-transferases(GSTs) are critical in maintaining redox homeostasis in plants, especially in relation to abiotic and biotic stress responses. However, the function of GSTs in resisting CBC remains unclear. Here, citrus glutathione S-transferases were investigated applying a genome-wide approach. In total, 69 CsGSTs belonging to seven classes were identified, and the phylogeny, chromosomal distribution, gene structures and conserved motifs were analyzed. Several CsGSTs responded to Xcc infection, as observed in the upregulation of CsGSTF1 and CsGSTU18 in the CBC-sensitive ‘Wanjincheng' variety but not in the resistant ‘Kumquat' variety. CsGSTF1 and CsGSTU18 were localized at the cytoplasm. Transient overexpression of CsGSTF1 and CsGSTU18 mediated reactive oxygen species(ROS) scavenging, whereas the virus-induced gene silencing(VIGS) of CsGSTF1 and CsGSTU18 caused strong CBC resistance and ROS burst. The present study investigated the characterization of citrus GST gene family, and discovered that CsGSTF1 and CsGSTU18 negatively contributed to CBC through modulating ROS homeostasis. These findings emphasize the significance of GSTs in infection resistance in plants.