The pepper extracellular xyloglucan-specific endo-ß-1,4-glucanase inhibitor protein gene, CaXEGIP1, is required for plant cell death and defense responses.

Plants produce various proteinaceous inhibitors to protect themselves against microbial pathogen attack. A xyloglucan-specific endo-ß-1,4-glucanase inhibitor1 gene, CaXEGIP1, was isolated and functionally characterized in pepper (Capsicum annuum) plants. CaXEGIP1 was rapidly and strongly induced in pepper leaves infected with avirulent Xanthomonas campestris pv vesicatoria, and purified CaXEGIP1 protein significantly inhibited the hydrolytic activity of the glycoside hydrolase74 family xyloglucan-specific endo-ß-1,4-glucanase from Clostridium thermocellum. Soluble-modified green fluorescent protein-tagged CaXEGIP1 proteins were mainly localized to the apoplast of onion (Allium cepa) epidermal cells. Agrobacterium tumefaciens-mediated overexpression of CaXEGIP1 triggered pathogen-independent, spontaneous cell death in pepper and Nicotiana benthamiana leaves. CaXEGIP1 silencing in pepper conferred enhanced susceptibility to virulent and avirulent X. campestris pv vesicatoria, accompanied by a compromised hypersensitive response and lowered expression of defense-related genes. Overexpression of dexamethasone:CaXEGIP1 in Arabidopsis (Arabidopsis thaliana) enhanced resistance to Hyaloperonospora arabidopsidis infection. Comparative histochemical and proteomic analyses revealed that CaXEGIP1 overexpression induced a spontaneous cell death response and also increased the expression of some defense-related proteins in transgenic Arabidopsis leaves. This response was also accompanied by cell wall thickening and darkening. Together, these results suggest that pathogen-inducible CaXEGIP1 positively regulates cell death-mediated defense responses in plants.

Detection of the plant pathogenic bacterium Xanthomonas campestris pv. Campestris in seed extracts of Brassica sp. Applying fluorescent antibodies and flow cytometry.

BACKGROUND: Xanthomonas campestris pv. campestris (Xcc) is a seed-transmitted plant pathogenic bacterium that causes black rot of crucifers. Seed lots and plants are screened for contamination with this pathogen using plating or serological assays. These methods, however, are time consuming and not very sensitive, respectively. Therefore, flow cytometry (FCM) was evaluated as a tool for the rapid detection and quantification of Xcc cells labeled with a mixture of specific fluorescein isothicyanate (FITC)-monoclonal antibodies (mAb) in pure culture, in mixed cultures of Xcc with either the common saprophyte Pseudomonas fluorescens (Psf) or a nonpathogenic X. campestris isolate (Xc), and in crude seed extracts. METHODS: The mAb 18G12, conjugated with FITC, was tested at dilutions of 1:50, 1:100, 1:200, and 1:400. For mixed suspensions of Xcc and Psf, mAb 18G12 was used at a dilution of 1:100. The combination of mAbs 18G12, 2F4, and 20H6, all conjugated with FITC, was used at a dilution of 1:100 for the detection and quantification of Xcc cells in mixed suspensions containing Xcc and Xc and in crude seed extracts. The analyses were performed with a Coulter EPICS XL-MCL flow cytometer, at low flow rate during 2 min. RESULTS: Using FCM, Xcc cells labeled with FITC-conjugated mAbs (18G12, 2F4, and 20H6) were detected and quantified rapidly at low numbers, i.e., 10(3) colony-forming units per milliliter in pure and in mixed cultures with Psf. The presence of the nonpathogenic Xc in the seed extracts did not interfere with the FCM results. Xcc cells were distinguished from the cells of other organisms and from small particles present in the seed extract based on the high-intensity fluorescence of the labeled cells. CONCLUSION: The application of FCM in combination with FITC-conjugated mAbs appears to be a promising technique for the detection and quantification of Xcc cells in seed extracts of crucifers.

An approach to obtain specific polyclonal antisera to Xanthomonas campestris pv. cyamopsidis and its potential application in indexing of infected seeds of guar.

Clusterbean seed health testing is warranted since the pathogen (Xanthomonas campestris pv. cyamopsidis (Xccy)) is seed-borne and seed-transmitted. A polyclonal antibody was developed in rabbit via subcutaneous and intramuscular injections and characterized for sensitivity, specificity and its applicability to ELISA which: (i) was sensitive in detecting as few as 102 cells ml - 1 at a titre of 1: 4000; (ii) was specific, since it reacted only with Xccy and not with other xanthomonads; (iii) reacted both with Xccy cells and culture filtrate, indicating that the antigenic determinant is a secretory component; (iv) was applicable and reliable in seed health testing since it reacted only with infected seeds and plant materials and not with healthy seeds and (v) a purified fraction of antibody was virulent-specific since heat-denatured and avirulent isolates were not detected. The ELISA thus developed is highly reproducible and therefore suitable for the evaluation of the potential disease status of seeds and plant health, which is appropriate for routine seed health testing.