Guo Zejian

Leucine zipper like structure in rice WRKY89 enhances its affinity for binding with W box elements

WRKY proteins are transcriptional regulators involved in plant responses to biotic and abiotic stresses, metabolisms, and developmental processes. In the present study, we isolated a WRKY cDNA, OsWRKY89 from a rice cDNA library. The deduced polypeptide contains 263 amino acid residues with a potential leucine zipper structure in its N-terminus, sharing low identity with other known WRKY members. OsWRKY89 and three deletion derivatives from its N-terminal were expressed in high levels in Escherichia coli as a C-terminally six-histidine-tagged fusion protein, and purified by employing one-step affinity chromatography on a Ni-NTA column. The recombinant OsWRKY89 protein was found to bind specially to sequences harboring W box cis elements by using electrophoretic mobility shift assays. This binding activity was decreased significantly by deletion of the leucine zipper-like structure in the N-terminal of Os-WRKY89. Using a yeast two-hybrid assay system, we found that the leucine zipper motif of OsWRKY89 was involved in the protein-protein interaction. Further deletion to remove partial WRKY domain abolished completely the interaction between the expressed protein and the W boxes, indicating that the WRKY domain is essential to the DNA-binding. These data strongly suggest that the leucine zipper-like motif of OsWRKY89 plays a significant role in the protein-protein and DNA-protein interactions.WRKY proteins are transcriptional regulators involved in plant responses to biotic and abiotic stresses, metabolisms, and developmental processes. In the present study, we isolated a WRKY cDNA, OsWRKY89 from a rice cDNA library. The deduced polypeptide contains 263 amino acid residues with a potential leucine zipper structure in its N-terminus, sharing low identity with other known WRKY members. OsWRKY89 and three deletion derivatives from its N-terminal were expressed in high levels in Escherichia coli as a C-terminally six-histidine-tagged fusion protein, and purified by employing one-step affinity chromatography on a Ni-NTA column. The recombinant OsWRKY89 protein was found to bind specially to sequences harboring W box cis elements by using electrophoretic mobility shift assays. This binding activity was decreased significantly by deletion of the leucine zipper-like structure in the N-terminal of Os-WRKY89. Using a yeast two-hybrid assay system, we found that the leucine zipper motif of OsWRKY89 was involved in the protein-protein interaction. Further deletion to remove partial WRKY domain abolished completely the interaction between the expressed protein and the W boxes, indicating that the WRKY domain is essential to the DNA-binding. These data strongly suggest that the leucine zipper-like motif of OsWRKY89 plays a significant role in the protein-protein and DNA-protein interactions.

Study on tobacco disease resistances mediated by the elicitor gene cryptogein from Phytophthora cryptogea

The cryptogein ( Crypt ) gene was obtained by PCR amplification of genomic DNA from Phytophthora cryptogea and confirmed by DNA sequencing. A promoter of the rice phenylalanine ammonia-lyase ( PAL ) gene was used to regulate the expression of Crypt , because this promoter has a low level of constitutive expression, is strongly induced by pathogen infection and is expressed in epidermal tissues. These promoter characteristics may be suitable for potential inhibition of pathogen attack on epidermal tissues. For functional interaction of Crypt with its outer plasma membrane binding sites, Crypt was led by a signal sequence of the extracellular pathogenesis-related protein (PR1b) of tobacco ( Nicotiana tabacum ). The fused gene was constructed into a binary vector and the final plasmid ( CHF-PAL::Crypt ) was transformed into tobacco using the Agrobacterium -mediated transformation method. Twenty-two lines of transformants were obtained from selection medium containing 100 mg/l of kanamycin. Results from PCR amplification and Southern blot analysis demonstrated that Crypt was integrated into the tobacco genome. In the assay of pathogen challenge, nearly two-thirds (68.2%) of the transgenic plants showed significantly enhanced resistance against black shank fungal ( Phytophthora parasitica var. nicotianae ), brown spot fungal ( Alternaria alternata ) and wild fire bacterial ( Pseudomonas syringae pv. tabaci ) diseases. This observation indicates that low-level constitutive expression of Crypt gene in tobacco could have potential use in generating broad-spectrum disease-resistant plants.

Isolation and transformation of Trichoderma viride protoplasts

The conditions for protoplast isolation and regeneration from Trichoderma viride were studied. Protoplasts were optimally isolated when mycelia of T. viride that had been cultured for 24 h were digested with 4 mg/ml Glucanex in phosphate buffer (pH 6.98) for 4 h at 30°C, resulting in a protoplast yield of 4.7×10 7 cfu/mg. The maximum regeneration ratio (14.5%) was obtained in mycelia culture medium containing 0.3 mol/l KCl and 0.3 mol/l inositol. In addition, a plasmid pCSSNCC1 carrying a hygromycin resistance gene and an elicitor-producing gene was transformed into T. viride protoplasts, with an efficiency of 1–2 transformants/μg DNA. The hygromycin-resistant transformants were determined by PCR and the elicitor protein was detected by ELISA. The results indicate that the elicitor protein was expressed stably in T. viride .