Morphological and molecular diversity of Sclerotinia sclerotiorum infecting Indian mustard.

Fourteen isolates of Sclerotinia sclerotiorum were collected from different locations of mustard growing regions of India and were studied for cultural, morphological and molecular variability at CCS HAU, Hisar. Variability was observed for colony colour, type of growth, diameter of mycelial growth, sclerotia initiation, number and pattern of sclerotia formation among the isolates. Mycelial growth and sclerotia initiation were faster in Bhiwani isolate as compared to others. Bhiwani isolate was found to be the most diverse and had least similarity with Chhanibari isolate on the basis of molecular variability. Hence, morphological and cultural variability observed in the present investigation is by and large strongly correlated to molecular marker based variability.

The rice OsSAG12-2 gene codes for a functional protease that negatively regulates stress-induced cell death.

Senescence is the final stage of plant development. Although expression of most of the genes is suppressed during senescence, a set of genes referred as senescence-associated genes (SAGs) is induced. Arabidopsis thaliana SAG12 (AtSAG12) is one such gene that has been mostly studied for its strict association with senescence. AtSAG12 encodes a papain-like cysteine protease, expressed predominantly in senescence-associated vacuoles. Rice genome contains multiple AtSAG12 homologues (OsSAGs). OsSAG12-1, the closest structural homologue of AtSAG12, is a negative regulator of developmental and stress-induced cell death. Proteolytic activity has not been established for any SAG12 homologues in vitro. Here, we report that OsSAG12-2, the second structural homologue of AtSAG12 from rice, codes for a functional proteolytic enzyme. The recombinant OsSAG12-2 protein produced in Escherichia coli undergoes autolysis to generate a functional protease. The matured OsSAG12-2 protein shows 27 percent trypsin-equivalent proteolytic activity on azocasein substrate. Dark-induced senescence activates OsSAG12-2 expression. Down-regulation of OsSAG12-2 in the transgenic artificial miRNA lines results in enhanced salt- and UV-induced cell death, even though it does not affect cell viability in the stress-free condition. Our results show that OsSAG12-2 codes for a functional protease that negatively regulates stress-induced cell death in rice.

Over-expression of Arabidopsis thaliana SFD1/GLY1, the gene encoding plastid localized glycerol-3-phosphate dehydrogenase, increases plastidic lipid content in transgenic rice plants.

Lipids are the major constituents of all membranous structures in plants. Plants possess two pathways for lipid biosynthesis: the prokaryotic pathway (i.e., plastidic pathway) and the eukaryotic pathway (i.e., endoplasmic-reticulum (ER) pathway). Whereas some plants synthesize galactolipids from diacylglycerol assembled in the plastid, others, including rice, derive their galactolipids from diacylglycerols assembled by the eukaryotic pathway. Arabidopsis thaliana glycerol-3-phosphate dehydrogenase (G3pDH), coded by SUPPRESSOR OF FATTY ACID DESATURASE 1 (SFD1; alias GLY1) gene, catalyzes the formation of glycerol 3-phosphate (G3p), the backbone of many membrane lipids. Here SFD1 was introduced to rice as a transgene. Arabidopsis SFD1 localizes in rice plastids and its over-expression increases plastidic membrane lipid content in transgenic rice plants without any major impact on ER lipids. The results suggest that over-expression of plastidic G3pDH enhances biosynthesis of plastid-localized lipids in rice. Lipid composition in the transgenic plants is consistent with increased phosphatidylglycerol synthesis in the plastid and increased galactolipid synthesis from diacylglycerol produced via the ER pathway. The transgenic plants show a higher photosynthetic assimilation rate, suggesting a possible application of this finding in crop improvement.

The Arabidopsis thaliana At4g13040 gene, a unique member of the AP2/EREBP family, is a positive regulator for salicylic acid accumulation and basal defense against bacterial pathogens.

The Arabidopsis genome contains a large number of putative transcription factors, containing a DNA binding domain similar to APETALA2/ethylene response element binding protein (AP2/EREBP), for most of which a function is not known. Phylogenetic analysis divides the Apetala 2 (AP2) super-family into 5 major groups: AP2, RAV, ethylene response factor (ERF), dehydration response element binding protein (DREB) and At4g13040. Similar to ERF and DREB, the At4g13040 protein contains only one AP2 domain; however, its structural uniqueness places it into a distinct group. In this article, we report that At4g13040 (referred herein as Apetala 2 family protein involved in SA mediated disease defense 1 - APD1) is an important regulator for SA mediated plant defense. The APD1 gene is upregulated upon pathogen inoculation, exogenous SA application and in the mutant that constitutively activates SA signaling. The T-DNA insertion lines (inserted in the APD1 promoter), which fail to induce expression upon pathogen inoculation, are compromised for resistance against virulent bacterial pathogens and show reduced induction of pathogenesis related 1 gene. Our results suggest that APD1 functions downstream of PAD4 in Arabidopsis and promotes pathogen-induced SA accumulation. Exogenous SA application completely restores the loss-of-resistance phenotype of the apd1 mutant. Thus, APD1 is a positive regulator of disease defense that functions upstream of SA accumulation.

Down-regulation of OsSAG12-1 results in enhanced senescence and pathogen-induced cell death in transgenic rice plants.

Senescence is a highly regulated process accompanied by changes in gene expression. While the mRNA levels of most genes decline, the mRNA levels of specific genes (senescence associated genes, SAGs) increase during senescence. Arabidopsis SAG12 (AtSAG12) gene codes for papain-like cysteine protease. The promoter of AtSAG12 is SA-responsive and reported to be useful to delay senescence by expressing cytokinin biosynthesis gene isopentenyltransferase specifically during senescence in several plants including Arabidopsis, lettuce and rice. The physiological role of AtSAG12 is not known; the homozygous atsag12 mutant neither fails to develop senescenceassociated vacuoles nor shows any morphological phenotype. Through BLAST search using AtSAG12 amino acid sequences as query, we identified a few putative homologues from rice genome (OsSAGs; Oryza sativa SAGs). OsSAG12-1 is the closest homologue of AtSAG12 with 64% similar amino acid composition. Expression of OsSAG12-1 is induced during senescence and pathogen-induced cell death. To evaluate the possible role of OsSAG12-1 we generated RNAi transgenic lines in Japonica rice cultivar TP309. The transgenic lines developed early senescence at varying levels and showed enhanced cell death when inoculated with bacterial pathogen Xanthomonas oryzae pv.oryzae. Our results suggest that OsSAG12-1 is a negative regulator of cell death in rice.