Functional analysis and expressional characterization of rice ankyrin repeat-containing protein, OsPIANK1, in basal defense against Magnaporthe oryzae attack.

The ankyrin repeat-containing protein gene OsPIANK1 (AK068021) in rice (Oryza sativa L.) was previously shown to be upregulated following infection with the rice leaf blight pathogen Xanthomonas oryzae pv oryzae (Xoo). In this study, we further characterized the role of OsPIANK1 in basal defense against Magnaporthe oryzae (M.oryzae) by 5' deletion analysis of its promoter and overexpression of the gene. The promoter of OsPIANK1 with 1,985 bps in length was sufficient to induce the OsPIANK1 response to inoculation with M.oryzae and to exogenous application of methyl jasmonate (MeJA) or salicylic acid (SA), but not to exogenous application of abscisic acid (ABA). A TCA-element present in the region between -563 bp and -249 bp may be responsible for the OsPIANK1 response to both M.oryzae infection and exogenous SA application. The JERE box, CGTCA-box, and two MYB binding sites locating in the region between -1985 bp and -907 bp may be responsible for the response of OsPIANK1 to exogenous MeJA. OsPIANK1 expression was upregulated after inoculation with M.oryzae and after treatment with exogenous SA and MeJA. Overexpression of OsPIANK1 enhanced resistance of rice to M.oryzae, although it did not confer complete resistance. The enhanced resistance to M.oryzae was accompanied by enhanced transcriptional expression of SA- and JA-dependent genes such as NH1, WKRY13, PAL, AOS2, PR1b, and PR5. This evidence suggests that OsPIANK1 acted as a positive regulator in rice basal defense mediated by SA- and JA-signaling pathways

Over-Expression of Rice CBS Domain Containing Protein, OsCBSX3, Confers Rice Resistance to Magnaporthe oryzae Inoculation

Cystathionine β-synthase (CBS) domain containing proteins (CDCPs) constitute a big family in plants and some members in this family have been implicated in a variety of biological processes, but the precise functions and the underlying mechanism of the majority of this family in plant immunity remain to be elucidated. In the present study, a CBS domain containing protein gene, OsCBSX3, is functionally characterized in rice resistance against Magnaporthe oryzae (M. oryzae). By quantitative real-time PCR, transcripts of OsCBSX3 are up-regulated significantly by inoculation of M. oryzae and the exogenously applied salicylic acid (SA) and methyl jasmonate (MeJA). OsCBSX3 is exclusively localized to the plasma membrane by transient expression of OsCBSX3 fused to green fluorescent protein (GFP) through approach of Agrobacterium infiltration in Nicotiana benthamiana leaves. The plants of homozygous T3 transgenic rice lines of over-expressing OsCBSX3 exhibit significant enhanced resistance to M. oryzae inoculation, manifested by decreased disease symptoms, and inhibition of pathogen growth detected in DNA. Consistently, the over-expression of OsCBSX3 enhances the transcript levels of immunity associated marker genes including PR1a, PR1b, PR5, AOS2, PAL, NH1, and OsWRKY13 in plants inoculated with M. oryzae. These results suggest that OsCBSX3 acts as a positive regulator in resistance of rice to M. oryzae regulated by SA and JA-mediated signaling pathways synergistically

Differential gene expression in pepper (<i style="">Capsicum annuum</i>) exposed to UV-B

429-437In the present paper, complementary DNA-amplified fragment length polymorphism (cDNA-AFLP) was used to examine and identify differentially expressed genes in Capsicum annuum exposed to UV-B irradiation. Around 4000 transcript derived fragments (TDFs) were visualized and in total 183 TDFs were isolated, sequenced and analyzed by Blast 2 go. Among these TDFs, 84 of them showed homology to known genes. There were 43 TDFs showing up-regulated expression, 24 TDFs showing down-regulated expression and 27 TDFs showing both up-regulated and down-regulated expression, respectively. Some of these TDFs were found to be in response/related to UV-B stress, including carbonic anhydrase, calcium-dependent protein, thionin-like protein, bzip protein and so on. In particular, chlorophyll a/b binding protein (Capcab) responding to UV-B stress was cloned. It was concluded that Capcab could play a protective role in plant anti-UV-B and maintaining photosynthetic rate under UV-B stress

<i>OsPIANK1</i> promoter activation in response to inoculation with <i>M.oryzae</i> or exogenous application of MeJA, SA or ABA.

<p>The leaves were harvested at 6 days after inoculation with <i>M.oryzae</i> and 12 h after treatment with the phytohormones. The numbers over the bars indicate the increase in induction of GUS activity compared to the mock. Data represent the means ± SE from the leaf extracts collected from three experimental plant units. (**, *indicate significant differences between the untreated (mock) and treated plants at <i>P</i><0.05 and <i>P</i><0.01, respectively, as determined by the SNK test.).</p

Nuclear localization of <i>Os</i>PIANK1 protein in <i>N. benthamiana</i> leaves.

<p><i>Os</i>PIANK1–GFP exclusively localized in the nucleus of cells in <i>N. benthamiana</i> leaves. Only the green fluorescent (GFP) localized throughout the whole cells. Cells were detected for GFP fluorescence by fluorescence microscopy 48 h after agroinfiltration.</p

Nucleotide sequence of 5′ flanking promoter regions and stress-related sequence motifs putatively acting as <i>cis</i>-elements of the <i>OsPIANK1</i> gene.

<p>HSE: <i>cis</i>-acting element involved in heat stress responsiveness. CGTCA-box: <i>cis</i>-acting regulatory element involved in the MeJA-responsiveness. JERE: <i>cis</i>-acting regulatory element involved in the MeJA-responsiveness. ERE: ethylene-responsive element. DRE: <i>cis</i>-acting element involved in dehydration, low-temp, salt stresses. MBS: MYB binding site involved in drought-inducibility. GCC-box: ethylene-responsive element. ABRE: <i>cis</i>-acting element involved in the abscisic acid responsiveness. TCA-element: <i>cis</i>-acting element involved in salicylic acid responsiveness.</p

Expression profiles of <i>OsPIANK1</i> in response to inoculation with <i>M.oryzae</i> or exogenous application of SA and MeJA.

<p>Transcript levels of <i>OsPIANK1</i> were determined by quantitative real-time PCR. Total RNA was prepared from leaf tissues of 3-week-old seedlings at the times indicated. The expression level of untreated plants at each time was used as the control and assigned a value of 1. Relative expression levels were normalized using the expression of <i>actin</i>. Data represents the mean ± SE of three independent biological replicates. (**,* indicate significant differences between the treatment and control at <i>P</i><0.01 or <i>P</i><0.05, respectively, as determined by Student–Newman–Keuls (SNK) test.).</p

The <i>OsPIANK1</i> promoter activities in different organs and its activation in response to <i>M.oryzae</i>.

<p>(A) Histochemical analysis of GUS activity in different organs of <i>OsPIANK1pro-GUS</i> transgenic rice. (B) Histochemical analysis of GUS activity in <i>OsPIANK1pro-GUS</i> transgenic rice response to inoculation of <i>M.oryzae</i>. The leaves were harvested at 4, 5, and 6 days after inoculation.</p