Ectopic expression of DnaJ type-I protein homolog of Vigna aconitifolia (VaDJI) confers ABA insensitivity and multiple stress tolerance in transgenic tobacco plants.

Reduced crop productivity results from altered plant physiological processes caused by dysfunctional proteins due to environmental stressors. In this study, a novel DnaJ Type-I encoding gene, VaDJI having a zinc finger motif in its C-terminal domain was found to be induced early upon treatment with heat stress (within 5 min) in a heat tolerant genotype of Vigna aconitifolia RMO-40. VaDJI is induced by multiple stresses. In tobacco, ectopic expression of VaDJI reduced ABA sensitivity during seed germination and the early stages of seedling growth of transgenic tobacco plants. Concomitantly, it also improved the ability of transgenic tobacco plants to withstand drought stress by modulating the photosynthetic efficiency, with the transgenic plants having higher Fv/Fm ratios and reduced growth inhibition. Additionally, transgenic plants showed a reduced build-up of H2O2 and lower MDA levels and higher chlorophyll content during drought stress, which attenuated cell damage and reduced oxidative damage. An analysis using the qRT-PCR study demonstrated that VaDJI overexpression is associated with the expression of some ROS-detoxification-related genes and stress-marker genes that are often induced during drought stress responses. These findings suggest a hypothesis whereby VaDJI positively influences drought stress tolerance and ABA signalling in transgenic tobacco, and suggests that it is a potential gene for genetic improvement of drought and heat stress tolerance in crop plants.

Global SUMOylome Adjustments in Basal Defenses of Arabidopsis thaliana Involve Complex Interplay Between SMALL-UBIQUITIN LIKE MODIFIERs and the Negative Immune Regulator SUPPRESSOR OF rps4-RLD1.

Steady-state SUMOylome of a plant is adjusted locally during developmental transitions and more globally during stress exposures. We recently reported that basal immunity in Arabidopsis thaliana against Pseudomonas syringae pv tomato strain DC3000 (PstDC3000) is associated with strong enhancements in the net SUMOylome. Transcriptional upregulations of SUMO conjugases, suppression of protease, and increased SUMO translations accounted for this enhanced SUMOylation. Antagonistic roles of SUMO1/2 and SUMO3 isoforms further fine-tuned the SUMOylome adjustments, thus impacting defense amplitudes and immune outcomes. Loss of function of SUPPRESSOR OF rps4-RLD1 (SRFR1), a previously reported negative regulator of basal defenses, also caused constitutive increments in global SUMO-conjugates through similar modes. These suggest that SRFR1 plays a pivotal role in maintenance of SUMOylation homeostasis and its dynamic changes during immune elicitations. Here, we demonstrate that SRFR1 degradation kinetically precedes and likely provides the salicylic acid (SA) elevations necessary for the SUMOylome increments in basal defenses. We show that SRFR1 not only is a SUMOylation substrate but also interacts in planta with both SUMO1 and SUMO3. In sum1 or sum3 mutants, SRFR1 stabilities are reduced albeit by different modes. Whereas a srfr1 sum1 combination is lethal, the srfr1 sum3 plants retain developmental defects and enhanced immunity of the srfr1 parent. Together with increasing evidence of SUMOs self-regulating biochemical efficiencies of SUMOylation-machinery, we present their impositions on SRFR1 expression that in turn counter-modulates the SUMOylome. Overall, our investigations reveal multifaceted dynamics of regulated SUMOylome changes via SRFR1 in defense-developmental balance.

Pathogen-induced AdDjSKI of the wild peanut, Arachis diogoi, potentiates tolerance of multiple stresses in E. coli and tobacco.

A gene encoding a serine-rich DnaJIII protein called AdDjSKI that has a 4Fe-4S cluster domain was found to be differentially upregulated in the wild peanut, Arachis diogoi in its resistance responses against the late leaf spot causing fungal pathogen Phaeoisariopsis personata when compared with the cultivated peanut, Arachis hypogaea. AdDjSKI is induced in multiple stress conditions in A. diogoi. Recombinant E. coli cells expressing AdDjSKI showed better growth kinetics when compared with vector control cells under salinity, osmotic, acidic and alkaline stress conditions. Overexpression of this type three J-protein potentiates not only abiotic stress tolerance in Nicotiana tabacum var. Samsun, but also enhances its disease resistance against the phytopathogenic fungi Phytophthora parasitica pv nicotianae and Sclerotinia sclerotiorum. In the present study we show transcriptional upregulation of APX, Mn-SOD and HSP70 under heat stress and increased transcripts of PR genes in response to fungal infection. This transmembrane-domain-containing J protein displays punctate localization in chloroplasts. AdDjSKI appears to ensure proper folding of proteins associated with the photosynthetic machinery under stress.

A novel zinc-binding alcohol dehydrogenase 2 from Arachis diogoi, expressed in resistance responses against late leaf spot pathogen, induces cell death when transexpressed in tobacco.

A novel zinc-binding alcohol dehydrogenase 2 (AdZADH2) was significantly upregulated in a wild peanut, Arachis diogoi treated with conidia of late leaf spot (LLS) pathogen, Phaeoisariopsis personata. This upregulation was not observed in a comparative analysis of cultivated peanut, which is highly susceptible to LLS. This zinc-binding alcohol dehydrogenase possessed a Rossmann fold containing NADB domain in addition to the MDR domain present in all previously characterized plant ADH genes/proteins. Transient over-expression of AdZADH2 under an estradiol inducible promoter (XVE) resulted in hypersensitive response (HR)-like cell death in tobacco leaf. However, the same level of cell death was not observed when the domains were transiently expressed individually. Cell death observed in tobacco was associated with overexpression of cell death related proteins, antioxidative enzymes such as SOD, CAT and APX and pathogenesis-related (PR) proteins. In A. diogoi, AdZADH2 expression was significantly upregulated in response to the plant signaling hormones salicylic acid, methyl jasmonate, and sodium nitroprusside.

Ectopic Expression of an Atypical Hydrophobic Group 5 LEA Protein from Wild Peanut, Arachis diogoi Confers Abiotic Stress Tolerance in Tobacco.

Late embryogenesis abundant (LEA) proteins are a group of hydrophilic proteins, which accumulate in plants under varied stress conditions like drought, salinity, extreme temperatures and oxidative stress suggesting their role in the protection of plants against these stresses. A transcript derived fragment (TDF) corresponding to LEA gene, which got differentially expressed in wild peanut, Arachis diogoi against the late leaf spot pathogen, Phaeoisariopsis personata was used in this study. We have cloned its full length cDNA by RACE-PCR, which was designated as AdLEA. AdLEA belongs to the atypical Group 5C of LEA protein family as confirmed by sequence analysis. Group 5C LEA protein subfamily contains Pfam LEA_2 domain and is highly hydrophobic. In native conditions, expression of AdLEA was upregulated considerably upon hormonal and abiotic stress treatments emphasizing its role in abiotic stress tolerance. Subcellular localization studies showed that AdLEA protein is distributed in both nucleus and cytosol. Ectopic expression of AdLEA in tobacco resulted in enhanced tolerance of plants to dehydration, salinity and oxidative stress with the transgenic plants showing higher chlorophyll content and reduced lipid peroxidation as compared to wild type plants. Overexpressed AdLEA tobacco plants maintained better photosynthetic efficiency under drought conditions as demonstrated by chlorophyll fluorescence measurements. These plants showed enhanced transcript accumulation of some stress-responsive genes. Our study also elucidates that ROS levels were significantly reduced in leaves and stomatal guard cells of transgenic plants upon stress treatments. These results suggest that AdLEA confers multiple stress tolerance to plants, which make it a potential gene for genetic modification in plants.

Characterization of a vacuolar processing enzyme expressed in Arachis diogoi in resistance responses against late leaf spot pathogen, Phaeoisariopsis personata.

Vacuolar processing enzymes are cysteine proteases responsible for maturation of vacuolar proteins. They have been shown to possess caspase-1-like activity, mediate cell death and display increased activity during pathogen infections. A transcript derived fragment corresponding to VPE was found to be up-regulated in a cDNA-AFLP analysis of host responses of a wild peanut, Arachis diogoi upon challenge from the late leaf spot pathogen Phaeoisariopsis personata, which was subsequently validated by q-PCR in a time course analysis, where susceptible peanut did not show its upregulation. In transient conditional and constitutive expression studies in tobacco leaves using agroinfiltration, we have observed that expression of AdVPE was associated with hypersensitive response (HR) like cell death. AdVPE expression was found to be high at 24 h post estradiol application and this was associated with the enhanced co-expression of molecular markers of HR cell death genes and genes for pathogenesis related proteins indicating that AdVPE positively regulates defense responses and its estradiol induced expression is sufficient for HR-like cell death in tobacco. We found that AdVPE expression was very strongly induced in response to sodium nitroprusside, which indicates its involvement in stress signaling. Induced expression of AdVPE in response to jasmonic acid and ethylene also indicates its involvement in an interconnected network of signaling. Transgenic tobacco plants ectopically expressing AdVPE exhibited enhanced resistance against Phytophthora parasitica var. nicotianae, Alternaria alternata var.  nicotianae and Rhizoctonia solani. To our knowledge, this is the first report on the heterologous expression of a pathogen induced VPE enhancing resistance to fungal pathogens with cell death phenomenon under transient expression.

Identification of heat-related ESTs in moth bean through suppression subtraction hybridization.

Moth bean (Vigna aconitifolia (Jacq.) Marechal), an important grain-legume crop grown in hot desert regions of Thar, under scorching sun rays, was investigated for heat tolerance at molecular level. In the present study, we constructed a forward suppression subtractive hybridization (SSH) cDNA library of heat tolerant genotype RMO-40 to identify genes expressing under delayed response to elevated temperature. Heat induction was carried out by exposing 14-day-old seedlings to elevated temperature of 42 °C for 30 min. A total of 125 unigenes (33 contigs and 92 singletons) were derived by cluster assembly and sequence alignment of 200 ESTs; out of 125 unigenes, 21 (16 %) were found to be novel to moth bean. Gene ontology functional classification terms were retrieved for 98 (78.4 %) unigenes of which 73 (58.4 %) ESTs were functionally annotated (GO consensus) where 19 unigenes were annotated with 11 enzyme commission (EC) codes and were mapped to 25 different KEGG pathways. We have identified a majority of heat-shock proteins (constituting 35 % of the present library) aiding heat stress tolerance to moth bean. An expression level of 22 ESTs generated from the above SSH cDNA library was studied through semiquantitative RT-PCR assay simultaneously under 5 and 30 min of heat stress at 42 °C.

Construction and analysis of an SSH cDNA library of early heat-induced genes of Vigna aconitifolia variety RMO-40.

Moth bean ( Vigna aconitifolia (Jacq.) Marechal) is an important grain legume crop grown in rain fed areas of hot desert regions of Thar, India, under scorching sun rays with very little supplementation of water. An SSH cDNA library was generated from leaf tissues of V. aconitifolia var. RMO-40 exposed to an elevated temperature of 42 °C for 5 min to identify early-induced genes. A total of 488 unigenes (114 contigs and 374 singletons) were derived by cluster assembly and sequence alignment of 738 ESTs; out of 206 ESTs (28%) of unknown proteins, 160 ESTs (14%) were found to be novel to moth bean. Only 578 ESTs (78%) showed significant BLASTX similarity (<1 × 10(-6)) in the NCBI non-redundant database. Gene ontology functional classification terms were retrieved for 479 (65%) sequences, and 339 sequences were annotated with 165 EC codes and mapped to 68 different KEGG pathways. Four hundred and fifty-two ESTs were further annotated with InterProScan (IPS), and no IPS was assigned to 153 ESTs. In addition, the expression level of 27 ESTs in response to heat stress was evaluated through semiquantitative RT-PCR assay. Approximately 20 different signaling genes and 16 different transcription factors have been shown to be associated with heat stress in moth bean for the first time.