Changes in redox status are a conspicuous feature of immune responses in a variety of eukaryotes, but the associated signalling mechanisms are not well understood. In plants, attempted microbial infection triggers the rapid synthesis of nitric oxide and a parallel accumulation of reactive oxygen intermediates, the latter generated by NADPH oxidases related to those responsible for the pathogen-activated respiratory burst in phagocytes. Both nitric oxide and reactive oxygen intermediates have been implicated in controlling the hypersensitive response, a programmed execution of plant cells at sites of attempted infection. However, the molecular mechanisms that underpin their function and coordinate their synthesis are unknown. Here we show genetic evidence that increases in cysteine thiols modified using nitric oxide, termed S-nitrosothiols, facilitate the hypersensitive response in the absence of the cell death agonist salicylic acid and the synthesis of reactive oxygen intermediates. Surprisingly, when concentrations of S-nitrosothiols were high, nitric oxide function also governed a negative feedback loop limiting the hypersensitive response, mediated by S-nitrosylation of the NADPH oxidase, AtRBOHD, at Cys 890, abolishing its ability to synthesize reactive oxygen intermediates. Accordingly, mutation of Cys 890 compromised S-nitrosothiol-mediated control of AtRBOHD activity, perturbing the magnitude of cell death development. This cysteine is evolutionarily conserved and specifically S-nitrosylated in both human and fly NADPH oxidase, suggesting that this mechanism may govern immune responses in both plants and animals.

Kaposi sarcoma-associated herpesvirus (KSHV) ORF57, also known as Mta (mRNA transcript accumulation), enhances viral intron-less transcript accumulation and promotes splicing of intron-containing viral RNA transcripts. In this study, we identified KSHV PAN, a long non-coding polyadenylated nuclear RNA as a main target of ORF57 by a genome-wide CLIP (cross-linking and immunoprecipitation) approach. KSHV genome lacking ORF57 expresses only a minimal amount of PAN. In cotransfection experiments, ORF57 alone increased PAN expression by 20-30-fold when compared to vector control. This accumulation function of ORF57 was dependent on a structured RNA element in the 5' PAN, named MRE (Mta responsive element), but not much so on an ENE (expression and nuclear retention element) in the 3' PAN previously reported by other studies. We showed that the major function of the 5' PAN MRE is increasing the RNA half-life of PAN in the presence of ORF57. Further mutational analyses revealed a core motif consisting of 9 nucleotides in the MRE-II , which is responsible for ORF57 interaction and function. The 9-nt core in the MRE-II also binds cellular PABPC1, but not the E1B-AP5 which binds another region of the MRE-II. In addition, we found that PAN RNA is partially exportable in the presence of ORF57. Together, our data provide compelling evidence as to how ORF57 functions to accumulate a non-coding viral RNA in the course of virus lytic infection.

Plant growth and development are regulated through coordinated interactions between light and phytohormones. Here, we demonstrate that a dark-induced small G protein, pea Pra2, regulates a variant cytochrome P450 that catalyzes C-2 hydroxylation in brassinosteroid biosynthesis. The cytochrome P450 is dark-induced and predominantly expressed in the rapidly elongating zone of etiolated pea epicotyls, where Pra2 is also most abundant. Transgenic plants with reduced Pra2 exhibit a dark-specific dwarfism, which is completely rescued by exogenous brassinolide. Overexpression of the cytochrome P450 results in enhanced hypocotyl growth even in the light, which phenocopies the etiolated hypocotyls. We therefore propose that Pra2 and its orthologs are molecular mediators for the cross-talk between light and brassinosteroids in the etiolation process in plants.

Kaposi's sarcoma-associated herpesvirus (KSHV) lytic infection increases the expression of viral and human interleukin-6 (vIL-6 and hIL-6, respectively), an important factor for cell growth and pathogenesis. Here, we report genome-wide analysis of viral RNA targets of KSHV ORF57 by a novel UV-cross-linking and immunoprecipitation (CLIP) assay. We identified 11 viral transcripts as putative ORF57 targets and demonstrate that vIL-6 mRNA is an authentic target of ORF57. Disrupting the ORF57 gene in the KSHV genome leads to inefficient expression of vIL-6. With transient transfection, the expression of vIL-6 could be enhanced greatly in the presence of ORF57 in a dose-dependent manner. We found that the open reading frame (ORF) region of vIL-6 RNA contains an MRE (MTA [ORF57]-responsive element) composed of two motifs, MRE-A and MRE-B, and binding of ORF57 to these two motifs stabilizes vIL-6 RNA and promotes vIL-6 translation. We demonstrate that vIL-6 MRE-B bears an miR-1293 binding site and that, mechanistically, ORF57 competes with miR-1293 for the same binding site to interact with vIL-6 RNA, thereby preventing vIL-6 RNA from association with the miR-1293-specified RNA-induced silencing complex (RISC). Consistent with this, ORF57 also interacts with an miR-608 binding site in the hIL-6 ORF and prevents miR-608 repression of hIL-6. Collectively, our results identify a novel function of ORF57 in being responsible for stabilization of viral and human IL-6 RNAs and the corresponding enhancement of RNA translation. In addition, our data provide the first evidence that a tumor virus may use a viral protein to interfere with microRNA (miRNA)-mediated repression of an miRNA target to induce cell proliferation and tumorigenesis during virus infection.Kaposi's sarcoma-associated herpesvirus (KSHV) or human herpesvirus 8 (HHV-8) is a lymphotropic DNA tumor virus, and when it infects B cells, it leads to development of body cavity-based B cell lymphoma and multicentric Castleman's disease (11,32). A hallmark indication of the infection is the increased expression of both viral interleukin-6 (vIL-6) and human interleukin-6 (hIL-6), which are used for clinical diagnosis and therapeutic targeting (2,16,31,33,36,37). Although the causes of increased expression of vIL-6 and hIL-6 during KSHV infection are not fully understood, increased IL-6 appears to be important to maintain cancer cell proliferation (6) and has been ascribed to transcriptional activation (6,8,9). Other studies indicate that various RNA elements in the 3Ј untranslated region (UTR) of hIL-6 mRNA contribute to rapid decay of hIL-6 mRNA and regulate hIL-6 expression primarily at the posttranscriptional level. These include an AU-rich element (ARE) interacting with AUF1 (38) and tristetraprolin (40), a non-ARE element interacting with Zc3h12a endonuclease (30), and microRNA (miRNA) seed matches binding let-7 (15) and miR-26 (17).An miRNA is a noncoding RNA that finely regulates the expression of target genes at the posttranscriptional level. In general, miRNAs ar...

Nitric oxide (NO) has been proposed to regulate a diverse array of activities during plant growth, development and immune function. S-nitrosylation, the addition of an NO moiety to a reactive cysteine thiol, to form an S-nitrosothiol (SNO), is emerging as a prototypic redox-based post-translational modification. An ARABIDOPSIS THALIANA S-NITROSOGLUTATHIONE (GSNO) REDUCTASE (AtGSNOR1) is thought to be the major regulator of total cellular SNO levels in this plant species. Here, we report on the impact of loss- and gain-of-function mutations in AtGSNOR1 upon plant growth and development. Loss of AtGSNOR1 function in atgsnor1-3 plants increased the number of initiated higher order axillary shoots that remain active, resulting in a loss of apical dominance relative to wild type. In addition atgsnor1-3 affected leaf shape, germination, 2,4-D sensitivity and reduced hypocotyl elongation in both light and dark grown seedlings. Silique size and seed production were also decreased in atgsnor1-3 plants and the latter was reduced in atgsnor1-1 plants, which overexpress AtGSNOR1. Overexpression of AtGSNOR1 slightly delayed flowering time in both long and short days, whereas atgsnor1-3 showed early flowering compared to wild type. In the atgsnor1-3 line, FLOWERING LOCUS C (FLC) expression was reduced, whereas transcription of CONSTANS (CO) was enhanced. Therefore, AtGSNOR1 may negatively regulate the autonomous and photoperiod flowering time pathways. Both overexpression and loss of AtGSNOR1 function also reduced primary root growth, while root hair development was increased in atgsnor1-1 and reduced in atgsnor1-3 plants. Collectively, our findings imply that AtGSNOR1 controls multiple genetic networks integral to plant growth and development.

Reversible protein phosphorylation, which is catalyzed by functionally coupled protein kinases and protein phosphatases, is a major signaling mechanism in eukaryotic cellular functions. The red and far-red light-absorbing phytochrome photoreceptors are light-regulated Ser/Thr-specific protein kinases that regulate diverse photomorphogenic processes in plants. Here

Nitric oxide (NO) is one of only a handful of gaseous signalling molecules. Its discovery as the endothelium-derived relaxing factor (EDRF) by Ignarro revolutionized how NO and cognate reactive nitrogen intermediates, which were previously considered to be toxic molecules, are viewed. NO is now emerging as a key signalling molecule in plants, where it orchestrates a plethora of cellular activities associated with growth, development, and environmental interactions. Prominent among these is its function in plant hypersensitive cell death and disease resistance. While a number of sources for NO biosynthesis have been proposed, robust and biologically relevant routes for NO production largely remain to be defined. To elaborate cell death during an incompatible plant-pathogen interaction NO functions in combination with reactive oxygen intermediates. Furthermore, NO has been shown to regulate the activity of metacaspases, evolutionary conserved proteases that may be intimately associated with pathogen-triggered cell death. NO is also thought to function in multiple modes of plant disease resistance by regulating, through S-nitrosylation, multiple nodes of the salicylic acid (SA) signalling pathway. These findings underscore the key role of NO in plant-pathogen interactions.

It now appears that photosynthetic prokaryotes and lower eukaryotes possess higher plant phytochrome-like proteins. In this work, a second phytochrome-like gene was isolated, in addition to the recently identified Cph1 phytochrome, from the Synechocystis sp. PCC 6803, and its gene product was characterized photochemically. The open reading frame sll0821 (designated cph2 in this work) has structural characteristics similar to those of the plant phytochromes and the Synechocystis Cph1 with high amino acid sequence homology in the N-terminal chromophore binding domain. The predicted Cph2 protein consists of 1276 amino acids with a calculated molecular mass of 145 kDa. Interestingly, the Cph2 protein has two putative chromophore binding domains, one around Cys-129 and the other around Cys-1022. The Cph2 was overexpressed in E. coli as an Intein/CBD (chitin binding domain) fusion and in vitro reconstituted with phycocyanobilin (PCB) or phytochromobilin (PPhiB). Both the Cph2-PCB and Cph2-PPhiB adducts showed the typical photochromic reversibility with the difference spectral maxima at 643/690 and 655/701 nm, respectively. The Cys-129 was confirmed to be the chromophore binding residue by in vitro mutagenesis and Zn(2+) fluorescence. The microenvironment of the chromophore in Cph2 seems to be similar to that in plant phytochromes. The cph2 gene expression was dark-induced and down-regulated to a basal level by light, like the cph1 gene. These observations suggest that Synechocystis species have multiple photosensory proteins, probably with distinct roles, as in higher plants.