Early detection of salt stress is vital for plant survival and growth. Still, the molecular processes controlling early salt stress perception and signaling are not fully understood. Here, we identified SALT-RESPONSIVE ERF1 (SERF1), a rice (Oryza sativa) transcription factor (TF) gene that shows a root-specific induction upon salt and hydrogen peroxide (H 2 O 2 ) treatment. Loss of SERF1 impairs the salt-inducible expression of genes encoding members of a mitogen-activated protein kinase (MAPK) cascade and salt tolerance-mediating TFs. Furthermore, we show that SERF1-dependent genes are H 2 O 2 responsive and demonstrate that SERF1 binds to the promoters of MAPK KINASE KINASE6 (MAP3K6), MAPK5, DEHYDRATION-RESPONSIVE ELEMENT BINDING2A (DREB2A), and ZINC FINGER PROTEIN179 (ZFP179) in vitro and in vivo. SERF1 also directly induces its own gene expression. In addition, SERF1 is a phosphorylation target of MAPK5, resulting in enhanced transcriptional activity of SERF1 toward its direct target genes. In agreement, plants deficient for SERF1 are more sensitive to salt stress compared with the wild type, while constitutive overexpression of SERF1 improves salinity tolerance. We propose that SERF1 amplifies the reactive oxygen species-activated MAPK cascade signal during the initial phase of salt stress and translates the salt-induced signal into an appropriate expressional response resulting in salt tolerance.

The OsCPK4 gene is a member of the complex gene family of calcium-dependent protein kinases in rice (Oryza sativa). Here, we report that OsCPK4 expression is induced by high salinity, drought, and the phytohormone abscisic acid. Moreover, a plasma membrane localization of OsCPK4 was observed by transient expression assays of green fluorescent protein-tagged OsCPK4 in onion (Allium cepa) epidermal cells. Overexpression of OsCPK4 in rice plants significantly enhances tolerance to salt and drought stress. Knockdown rice plants, however, are severely impaired in growth and development. Compared with control plants, OsCPK4 overexpressor plants exhibit stronger water-holding capability and reduced levels of membrane lipid peroxidation and electrolyte leakage under drought or salt stress conditions. Also, salt-treated OsCPK4 seedlings accumulate less Na in their roots. We carried out microarray analysis of transgenic rice overexpressing OsCPK4 and found that overexpression of OsCPK4 has a low impact on the rice transcriptome. Moreover, no genes were found to be commonly regulated by OsCPK4 in roots and leaves of rice plants. A significant number of genes involved in lipid metabolism and protection against oxidative stress appear to be up-regulated by OsCPK4 in roots of overexpressor plants. Meanwhile, OsCPK4 overexpression has no effect on the expression of well-characterized abiotic stress-associated transcriptional regulatory networks (i.e. ORYZA SATIVA DEHYDRATION-RESPONSIVE ELEMENT BINDING PROTEIN1 and ORYZA SATIVA No Apical Meristem, Arabidopsis Transcription Activation Factor1-2, Cup-Shaped Cotyledon6 genes) and LATE EMBRYOGENESIS ABUNDANT genes in their roots. Taken together, our data show that OsCPK4 functions as a positive regulator of the salt and drought stress responses in rice via the protection of cellular membranes from stress-induced oxidative damage.

SUMMARYIn mammals, lipid bodies play a key role during pathological and infectious diseases. However, our knowledge on the function of plant lipid bodies, apart from their role as the major site of lipid storage in seed tissues, remains limited. Here, we provide evidence that a calcium-dependent protein kinase (CPK) mediates pathogen resistance in Arabidopsis. AtCPK1 expression is rapidly induced by fungal elicitors. Loss-of-function mutants of AtCPK1 exhibit higher susceptibility to pathogen infection compared to wild-type plants. Conversely, overexpression of AtCPK1 leads to accumulation of salicylic acid (SA) and constitutive expression of SA-regulated defence and disease resistance genes, which, in turn, results in broad-spectrum protection against pathogen infection. Expression studies in mutants affected in SA-mediated defence responses revealed an interlocked feedback loop governing AtCPK1 expression and components of the SA-dependent signalling pathway. Moreover, we demonstrate the dual localization of AtCPK1 in lipid bodies and peroxisomes. Overall, our findings identify AtCPK1 as a component of the innate immune system of Arabidopsis plants.

Plants have evolved efficient defence mechanisms to defend themselves from pathogen attack. Although many studies have focused on the transcriptional regulation of defence responses, less is known about the involvement of microRNAs (miRNAs) as post-transcriptional regulators of gene expression in plant immunity. This work investigates miRNAs that are regulated by elicitors from the blast fungus Magnaporthe oryzae in rice (Oryza sativa). Small RNA libraries were constructed from rice tissues and subjected to high-throughput sequencing for the identification of elicitor-responsive miRNAs. Target gene expression was examined by microarray analysis. Transgenic lines were used for the analysis of miRNA functioning in disease resistance. Elicitor treatment is accompanied by dynamic alterations in the expression of a significant number of miRNAs, including new members of annotated miRNAs. Novel miRNAs from rice are proposed. We report a new rice miRNA, osa-miR7695, which negatively regulates an alternatively spliced transcript of OsNramp6 (Natural resistance-associated macrophage protein 6). This novel miRNA experienced natural and domestication selection events during evolution, and its overexpression in rice confers pathogen resistance. This study highlights an miRNA-mediated regulation of OsNramp6 in disease resistance, whilst illustrating the existence of a novel regulatory network that integrates miRNA function and mRNA processing in plant immunity.

Arbuscular mycorrhizal (AM) symbioses are mutualistic associations between soil fungi and most vascular plants. Their association benefits the host plant by improving nutrition, mainly phosphorus nutrition, and by providing increased capability to cope with adverse conditions. In this study, we investigated the transcriptional changes triggered in rice leaves as a result of AM symbiosis, focusing on the relevance of the plant defence response. We showed that root colonization by the AM fungus Glomus intraradices is accompanied by the systemic induction of genes that play a regulatory role in the host defence response, such as OsNPR1, OsAP2, OsEREBP and OsJAmyb. Genes involved in signal transduction processes (OsDUF26 and OsMPK6) and genes that function in calcium-mediated signalling processes (OsCBP, OsCaM and OsCML4) are also up-regulated in leaves of mycorrhizal rice plants in the absence of pathogen infection. In addition, the mycorrhizal rice plants exhibit a stronger induction of defence marker genes [i.e. pathogenesis-related (PR) genes] in their leaves in response to infection by the blast fungus Magnaporthe oryzae. Evidence indicates that mycorrhizal rice plants show enhanced resistance to the rice blast fungus. Overall, these results suggest that the protective effect of the AM symbiosis in rice plants relies on both the systemic activation of defence regulatory genes in the absence of pathogen challenge and the priming for stronger expression of defence effector genes during pathogen infection. The possible mechanisms involved in the mycorrhiza-induced resistance to M. oryzae infection are discussed.

Expression of pathogenesis-related (PR) genes is part of the plant's natural defense response against pathogen attack. The PRms gene encodes a fungal-inducible PR protein from maize. Here, we demonstrate that expression of PRms in transgenic rice confers broad-spectrum protection against pathogens, including fungal (Magnaporthe oryzae, Fusarium verticillioides, and Helminthosporium oryzae) and bacterial (Erwinia chrysanthemi) pathogens. The PRms-mediated disease resistance in rice plants is associated with an enhanced capacity to express and activate the natural plant defense mechanisms. Thus, PRms rice plants display a basal level of expression of endogenous defense genes in the absence of the pathogen. PRms plants also exhibit stronger and quicker defense responses during pathogen infection. We also have found that sucrose accumulates at higher levels in leaves of PRms plants. Sucrose responsiveness of rice defense genes correlates with the pathogen-responsive priming of their expression in PRms rice plants. Moreover, pretreatment of rice plants with sucrose enhances resistance to M. oryzae infection. Together, these results support a sucrose-mediated priming of defense responses in PRms rice plants which results in broad-spectrum disease resistance.

Cathepsin B is a lysosomal thiol proteinase that may have additional extralysosomal functions. To further our investigations on the structure, mode of biosynthesis, and intracellular sorting of this enzyme, we have determined the complete coding sequences for human and mouse preprocathepsin B by using cDNA clones isolated from human hepatoma and kidney phage libraries. The nucleotide sequences predict that the primary structure of preprocathepsin B contains 339 amino acids organized as follows: a 17-residue NH2-terminal prepeptide sequence followed by a 62-residue propeptide region, 254 residues in mature (single chain) cathepsin B, and a 6-residue extension at the COOH terminus. A comparison of procathepsin B sequences from three species (human, mouse, and rat) reveals that the homology between the propeptides is relatively conserved with a minimum of 68% sequence identity. In particular, two conserved sequences in the propeptide that may be functionally significant include a potential glycosylation site and the presence of a single cysteine at position 59. Comparative analysis of the three sequences also suggests that processing of procathepsin B is a multistep process, during which enzymatically active intermediate forms may be generated. The availability of the cDNA clones will facilitate the identification of possible active or inactive intermediate processive forms as well as studies on the transcriptional regulation of the cathepsin B gene.Cathepsin B is a member of a superfamily of structurally similar tissue proteinases having a catalytic unit of -25 kDa that contains an active center made up of side chains derived from a cysteine residue located in its NH2-terminal region and a more COOH-terminally located histidine residue (1). These thiol proteinases are structurally and functionally closely related to papain as well as to actinidin, both plant enzymes (2). Cathepsin B also shows significant amino acid sequence homology to the proteolytic domain of the cytosolic calciumdependent proteases (3), indicating that further evolutionary diversification has occurred within this proteolytic superfamily. Mature cathepsin B and the related thiol cathepsins H and L as well as a number of other exo-or endoproteinases have been localized to the lysosomes in various cells, indicating that these proteinases are involved in protein turnover (4).Recent biosynthetic studies in our laboratory have indicated that cathepsin B is derived in biosynthesis from a larger precursor form, or procathepsin B, which in its glycosylated state has a molecular size of -40 kDa (5). In isolated islets of Langerhans, this precursor form is either secreted into the medium or is slowly converted to material similar in size and immunological properties to mature cathepsin B and localized in lysosomal and secretion granule fractions; e.g., Docherty et al. (6) have identified both 31-kDa and 38-kDa cathepsin B-like proteins in purified secretion granules from a rat insulinoma and in normal rat islet granule fractions (7). A functional...

The nonexpressor of pathogenesis-related (PR) genes (NPR1) protein plays an important role in mediating defense responses activated by pathogens in Arabidopsis. In rice, a disease-resistance pathway similar to the Arabidopsis NPR1-mediated signaling pathway one has been described. Here, we show that constitutive expression of the Arabidopsis NPR1 (AtNPR1) gene in rice confers resistance against fungal and bacterial pathogens. AtNPR1 exerts its protective effects against fungal pathogens by priming the expression of salicylic acid (SA)-responsive endogenous genes, such as the PR1b, TLP (PR5), PR10, and PBZ1. However, expression of AtNPR1 in rice has negative effects on viral infections. The AtNPR1-expressing rice plants showed a higher susceptibility to infection by the Rice yellow mottle virus (RYMV) which correlated well with a misregulation of RYMV-responsive genes, including expression of the SA-regulated RNA-dependent RNA polymerase 1 gene (OsRDR1). Moreover, AtNPR1 negatively regulates the expression of genes playing a role in the plant response to salt and drought stress (rab21, salT, and dip1), which results in a higher sensitivity of AtNPR1 rice to the two types of abiotic stress. These observations suggest that AtNPR1 has both positive and negative regulatory roles in mediating defense responses against biotic and abiotic stresses.