Summary: A hot pepper (Capsicum annuum) cDNA clone encoding pathogenesis‐related protein 10 (CaPR‐10) was isolated by differential screening of a cDNA library prepared from pepper leaves inoculated with tobacco mosaic virus pathotype (TMV‐P₀).CaPR‐10transcripts were induced in the incompatible interaction with TMV‐P₀orXanthomonas campestrispv.vesicatoria(Xcv) but not induced in the compatible interaction. Characterization of enzymatic properties of CaPR‐10 indicated that the recombinant protein exhibits a ribonucleolytic activity against TMV RNA, as well as against pepper total RNA, and shows its putative antiviral activity in several conditions. The CaPR‐10 protein existed at very low level in leaf tissue but was dramatically induced as soon as plants were inoculated with TMV‐P₀, and this was correlated with the increase of its ribonucleolytic activity. Immunoblot analysis and pull‐down assays using proteins extracted from pepper leaves showed that TMV‐P₀inoculation led to the phosphorylation of CaPR‐10, a modification that should affect its capacity for RNase function. We present data that the induction and subsequent phosphorylation of CaPR‐10 increased its ribonucleolytic activity to cleave invading viral RNAs, and this activity should be important to its antiviral pathway during viral attackin vivo.

A mutant screening was carried out previously to look for new genes related to the Cucumber mosaic virus infection response in Arabidopsis. A Pumilio RNA binding protein-coding gene, Arabidopsis Pumilio RNA binding protein 5 (APUM5), was obtained from this screening.

Posttranscriptional/translational regulation of gene expression is mediated by diverse RNA binding proteins and plays an important role in development and defense processes. Among the RNA-binding proteins, the mammalian Pumilio RNA-binding family (Puf) acts as posttranscriptional and translational repressors. AnArabidopsisPuf mutant,apum5-D, was isolated during a T-DNA insertional mutant screen for mutants with reduced susceptibility toCucumber mosaic virus(CMV) infection. Interestingly, CMV RNA contained putative Pumilio-homology domain binding motifs in its 3′ untranslated region (UTR) and internal places in its genome. APUM5 directly bound to the 3′ UTR motifs and some internal binding motifs in CMV RNAs in vitro and in vivo. We showed that APUM5 acts as a translational repressor that regulates the 3′ UTR of CMV and affects CMV replication. This study uncovered a unique defense system thatArabidopsisAPUM5 specifically regulates CMV infection by the direct binding of CMV RNAs.

The transcription factor ATAF2, one of the plant specific NAC family genes, is known as repressor of pathogenesisrelated genes and responsive to the diverse defense-related hormones, pathogen infection, and wounding stress. Furthermore, it is important to consider that tryptophandependant IAA biosynthesis pathway can be activated by wounding and pathogen. We found that ATAF2pro::GUS reporter was induced upon indole-3-acetonitrile (IAN) treatments. And ataf2 mutant showed reduced sensitivity to IAN whereas 35S::ATAF2 plants showed hyper-sensitivity to IAN. IAN biosynthesis required nitrilase involved in the conversion of IAN to an auxin, indole-3-acetic acid (IAA). We found that the NIT2 gene was repressed in ataf2 knockout plants. Expression of both ATAF2 and NIT2 genes was induced by IAN treatment. Transgenic plants overexpressing ATAF2 showed up-regulated NIT2 expression. ATAF2 activated promoter of the NIT2 gene in Arabidopsis protoplasts. Electrophoretic mobility shift assay revealed that NIT2 promoter region from position -117 to -82 contains an ATAF2 binding site where an imperfect palindrome sequence was critical to the protein-DNA interaction. These findings indicate that ATAF2 regulates NIT2 gene expression via NIT2 promoter binding.

Tobacco stress-induced1 (Tsi1) is an ethylene-responsive-element binding protein/APETALA2-type transcription factor that plays an important role in both biotic and abiotic stress signaling pathways. We show that Tsi1-interacting protein1 (Tsip1), a DnaJ-type Zn finger protein, interacts with Tsi1 in vitro and in yeast (Saccharomyces cerevisiae). The transcript level of Tsip1 in tobacco (Nicotiana tabacum) increased upon treatment with salicylic acid (SA), ethylene, gibberellic acid, NaCl, and virus challenge. Tsip1 appeared to be physically associated with the chloroplast surface but dissociated from it after SA treatment. Tsip1 colocalized and coimmunoprecipitated with Tsi1 in plant cells following SA treatment. Tsip1 expression increased Tsi1-mediated transcription and was able to functionally compensate for loss of the Tsi1 transcriptional activation domain through a direct interaction with Tsi1. Transgenic plants simultaneously coexpressing Tsi1 and Tsip1 displayed stronger pathogen resistance and salt tolerance than did transgenic plants expressing either Tsi1 or Tsip1 alone. Concurrent with this, the expression of a subset of stress-related genes was induced in a cooperative manner in Tsi1/Tsip1 transgenic plants. These results together implied that Tsi1 recruits Tsip1 to the promoters of stress-related genes to potentiate Tsi1-mediated transcriptional activation.