High-throughput screening system of citrus bacterial cankerassociated transcription factors and its application to the regulation of citrus canker resistance

One of the main diseases that adversely impacts the global citrus industry is citrus bacterial canker(CBC),caused by the bacteria Xanthomonas citri subsp.citri(Xcc).Response to CBC is a complex process,with both proteinDNA as well as protein–protein interactions for the regulatory network.To detect such interactions in CBC resistant regulation,a citrus high-throughput screening system with 203 CBC-inducible transcription factors(TFs),were developed.Screening the upstream regulators of target by yeast-one hybrid(Y1H)methods was also performed.A regulatory module of CBC resistance was identified based on this system.One TF(CsDOF5.8)was explored due to its interactions with the 1-kb promoter fragment of CsPrx25,a resistant gene of CBC involved in reactive oxygen species(ROS)homeostasis regulation.Electrophoretic mobility shift assay(EMSA),dual-LUC assays,as well as transient overexpression of CsDOF5.8,further validated the interactions and transcriptional regulation.The CsDOF5.8–CsPrx25 promoter interaction revealed a complex pathway that governs the regulation of CBC resistance via H2O2homeostasis.The high-throughput Y1H/Y2H screening system could be an efficient tool for studying regulatory pathways or network of CBC resistance regulation.In addition,it could highlight the potential of these candidate genes as targets for efforts to breed CBC-resistant citrus varieties.

Estimation of citrus canker lesion size using hyperspectral reflectance imaging

The Citrus industry has need for effective approaches to remove fruit with canker before they are shipped to selective international market such as the European Union.This research aims to determine the detectable size limit for cankerous lesions using hyperspectral imaging approaches.Previously developed multispectral algorithms using visible to near-infrared wavelengths,were used to segregate cankerous citrus fruits from other peel conditions(normal,greasy spot,insect damage,melanose,scab and wind scar).However,this previous work did not consider lesion size.A two-band ratio method with a simple threshold based classifier(ratio of reflectance at wavelengths 834 nm and 729 nm),which gave maximum overall classification accuracy of 95.7%,was selected for lesion size estimation in this study.The smallest size of cankerous lesion detected in terms of equivalent diameter was 1.66 mm.The effect of variation of threshold values and number of erosion cycles(applying morphological erosion multiple times to the image)on estimation of smallest detectable lesion was observed.It was found that small threshold values gave better canker classification accuracies,while exhibiting a lower overall classification accuracy.Meanwhile,higher threshold values portrayed the opposite tendency.The threshold value of 1.275 gave the optimum tradeoff between canker classification accuracy,overall classification accuracy and minimal lesion size detection.Increasing the number of erosion cycles reduced detection rates of smaller canker lesions,leading to the conclusion that a single erosion cycle gave the best size estimation results.The erosion kernel of the size 3 mm×3 mm was used during the exploration.

Production of Transgenic Anliucheng Sweet Orange(Citrus sinensis Osbeck) with Xa21 Gene for Potential Canker Resistance

Citrus canker, an epidemic quarantine disease caused by Xanthomonas axonopodis pv. citri, has brought a great damage in citrus production worldwide. Herein, a rice PRR （pattern recognition receptor） gene Xa21 together with GUS reporter gene and hygromycin phosphotransferase gene （HPT） was introduced into Anliucheng sweet orange （Citrus sinensis Osbeck） via Agrobacterium-mediated transformation of embryogenic callus. The transgenic calluses were screened on MT basal medium containing hygromycin （HYG） and detected by histochemical GUS staining. The transgenic plantlets were recovered through somatic embryogenesis pathway. The regenerated plantlets were accustomed to and maintained in the greenhouse. The transgene integration of recovered plantlets was identiifed by PCR and Southern blot hybridization. It showed that all the transgenic plantlets tested had undergone single copy integration, the expression of Xa21 in eight different transgenic lines detected by qRT-PCR can be divided into three grades, high for T5 and T6, middle for T4 and low for the rest. The tolerance to citrus canker disease of the three recovered transgenic lines T2, T4 and T6 was assessed by in vitro pin-puncture inoculation. The results showed that all the three transgenic lines conferred improved resistance to citrus canker bacterium infection and the T4 transgenic line displayed the highest resistance. The mechanism and feasibility of rice Xa21 in triggering innate immunity in citrus was brielfy discussed.

Occurrence Regularity and Integrated Control Technology of Canker in Citrus

Canker is a quarantine bacterial disease that seriously harms leaves,branches and fruits of citrus,leading to a decrease in the production and affecting the commodity and sales of citrus.Citrus canker has the characteristics of fast spreading speed and difficult radical cure.Through the identification of symptoms and a summary of occurrence regularity,the integrated prevention and control technology for citrus canker is described in this article,in order to achieve effective prevention and control and reduce prevention and control cost.

Preparation of Monoclonal Antibody Against PthA-NLS and Cloning of the Relative ScFv Gene

The present study aimed at the preparation of monoclonal antibody against the recombinant PthA-NLS and the isolation of the relative ScFv （single chain variable fragment） genes, providing the possibility to better understand the pathogenesis mechanism via PthA, and developing proper construct for future experimentation to obtain citrus plants resistant to canker disease by transformation and plant antibody techniques. The recombinant polypeptide PthA-NLS was injected into Balb/c mice to produce monoclonal antibody. Total RNA was isolated from the hybridoma cell line 3D10H2 which secreted anti- PthA-NLS McAb, and the variable region genes were amplified with specific primers by RT-PCR and SOE-PCR （splicing by overlap extension）, and then the ScFv gene was isolated. The recombinant ScFv gene was cloned into pGEM-T and pET32a（＋） vector. The later plasmid was transferred into E. coli BL21 （DE3） and the expression of the recombinant protein was induced. Three cell lines producing monoclonal antibody against PthA-NLS were acquired and named 1C8H1, 2D12B6, and 3D8A10. The recombinant ScFv gene of about 750 bp was constructed. The sequencing results showed that the ScFv gene consists of a 360 bp heavy chain, a 342 bp light chain, and a 45 bp linker region. The recombinant fusion ScFv protein was expressed by IPTG induction, and a 44.5 kDa of recombinant fusion protein was obtained. In conclusion, we obtained three cell lines stably producing monoclonal antibody specifically bound to PthA-NLS, and the relative ScFv gene was constructed and successfully expressed in E. coli. These results may play an important role in further understanding the pathogenesis mechanism and in the development of possible citrus resistant to canker disease by genetic transformation and plant antibiobody.

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.

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.

Cloning and Expression Analysis of Citrus Genes Cs GH3.1 and Cs GH3.6Responding to Xanthomonas axonopodis pv. citri Infection

To study the functions of the early auxin-responsive genes Cs GH3.1 and Cs GH3.6 in citrus resistance against canker disease, we cloned Cs GH3.1and Cs GH3.6 in ‘Newhall' Navel Orange(Citrus sinensis Osbeck). They are 1 797 bp and 1 887 bp and encode 598 and 629 amino acids, respectively. In vitro mature leaves from susceptible ‘Newhall' and resistant Calamondin(C. madurensis) were inoculated by a Xanthomonas axonopodis pv. citri(Xac) bacterial suspension, and expression of Cs GH3.1 and Cs GH3.6 in the two varieties were analyzed using quantitative real-time PCR(q RT-PCR). ‘Newhall' leaves were treated with different hormones for 3 days, inoculated by Xac bacterial suspension, and then the symptoms in these leaves were investigated. We used q RT-PCR to analyze the effect of different hormones on Cs GH3.1 and Cs GH3.6 expression in ‘Newhall'leaves. The expression levels of both Cs GH3.1 and Cs GH3.6 were significantly induced by Xac in ‘Newhall' leaves, compared with levels in Calamondin leaves. 1-naphthy acetic acid(NAA) increased the hypertrophy of infection sites in ‘Newhall' leaves, while naphthyl-phthalamic acid(NPA)had no visible effect on lesion development. NAA hormone greatly improved expression of Cs GH3.1 in ‘Newhall', but not Cs GH3.6. These results indicate that the auxin primary-response gene Cs GH3.1 plays an important role in citrus susceptibility to Xac.