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The wild plant species Calotropis procera (C. procera) has many potential applications and beneficial uses in medicine, industry and ornamental field. It also represents an excellent source of genes for drought and salt tolerance. Genes encoding proteins that contain the conserved universal stress protein (USP) domain are known to provide organisms like bacteria, archaea, fungi, protozoa and plants with the ability to respond to a plethora of environmental stresses. However, information on the possible occurrence of Usp in C. procera is not available. In this study, we uncovered and characterized a one-class A Usp-like (UspA-like, NCBI accession No. KC954274) gene in this medicinal plant from the de novo assembled genome contigs of the high-throughput sequencing dataset. A number of GenBank accessions for Usp sequences were blasted with the recovered de novo assembled contigs. Homology modelling of the deduced amino acids (NCBI accession No. AGT02387) was further carried out using Swiss-Model, accessible via the EXPASY. Superimposition of C. procera USPA-like full sequence model on Thermus thermophilus USP UniProt protein (PDB accession No. Q5SJV7) was constructed using RasMol and Deep-View programs. The functional domains of the novel USPA-like amino acids sequence were identified from the NCBI conserved domain database (CDD) that provide insights into sequence structure/function relationships, as well as domain models imported from a number of external source databases (Pfam, SMART, COG, PRK, TIGRFAM)., (Copyright © 2014 Académie des sciences. All rights reserved.)

Repetitive rounds of differential subtraction screening, followed by nucleotide sequence determination and northern-blot analysis, identified 84 salt-regulated (160 mM NaCl for 4 h) genes in Arabidopsis wild-type (Col-0 gl1) seedlings. Probes corresponding to these 84 genes and ACP1, RD22BP1, MYB2, STZ, and PAL were included in an analysis of salt responsive gene expression profiles in gl1 and the salt-hypersensitive mutant sos3. Six of 89 genes were expressed differentially in wild-type and sos3 seedlings; steady-state mRNA abundance of five genes (AD06C08/unknown, AD05E05/vegetative storage protein 2 [VSP2], AD05B11/S-adenosyl-L-Met:salicylic acid carboxyl methyltransferase [SAMT], AD03D05/cold regulated 6.6/inducible2 [COR6.6/KIN2], and salt tolerance zinc finger [STZ]) was induced and the abundance of one gene (AD05C10/circadian rhythm-RNA binding1 [CCR1]) was reduced in wild-type plants after salt treatment. The expression of CCR1, SAMT, COR6.6/KIN2, and STZ was higher in sos3 than in wild type, and VSP2 and AD06C08/unknown was lower in the mutant. Salt-induced expression of VSP2 in sos1 was similar to wild type, and AD06C08/unknown, CCR1, SAMT, COR6.6/KIN2, and STZ were similar to sos3. VSP2 is regulated presumably by SOS2/3 independent of SOS1, whereas the expression of the others is SOS1 dependent. AD06C08/unknown and VSP2 are postulated to be effectors of salt tolerance whereas CCR1, SAMT, COR6.6/KIN2, and STZ are determinants that must be negatively regulated during salt adaptation. The pivotal function of the SOS signal pathway to mediate ion homeostasis and salt tolerance implicates AD06C08/unknown, VSP2, SAMT, 6.6/KIN2, STZ, and CCR1 as determinates that are involved in salt adaptation.

The capacity of plants to counter the challenge of pathogenic fungal attack depends in part on the ability of plant defense proteins to overcome fungal resistance by being able to recognize and eradicate the invading fungi. Fungal genes that control resistance to plant defense proteins are therefore important determinants that define the range of fungi from which an induced defense protein can protect the plant. Resistance of the model fungus Saccharomyces cerevisiae to osmotin, a plant defense PR-5 protein, is strongly dependent on the natural polymorphism of the SSD1 gene. Expression of the SSD1-v allele afforded resistance to the antifungal protein. Conversely, yeast strains carrying the SSD1-d allele or a null ssd1Delta mutation displayed high sensitivity to osmotin. The SSD1-v protein mediates osmotin resistance in a cell wall-dependent manner. Deletion of SSD1-v or SSD1-d impeded sorting of the PIR proteins (osmotin-resistance factors) to the cell wall without affecting mRNA levels, indicating that SSD1 functions in post-transcriptional regulation of gene expression. The sensitivity of ssd1Delta cells to osmotin was only partially suppressed by over-accumulation of PIR proteins in the cell wall, suggesting an additional function for SSD1 in cell wall-mediated resistance. Accordingly, cells carrying a null ssd1 mutation also displayed aberrant cell-wall morphology and lower levels of alkali-insoluble cell-wall glucans. Therefore SSD1 is an important regulator of fungal cell-wall biogenesis and composition, including the deposition of PIR proteins which block the action of plant antifungal PR-5 proteins.

Griffonia simplicifolia II, an N-acetylglucosamine-specific legume lectin, has insecticidal activity when fed to the cowpea weevil, Callosobruchus maculatus (F.). A cDNA clone encoding G. simplicifolia II was isolated from a leaf cDNA library, sequenced, and expressed in a bacterial expression system. The recombinant protein exhibited N-acetylglucosamine-binding and insecticidal activity against cowpea weevil, indicating that glycosylation and multimeric structure are not required for these properties. These results support the hypothesis that genes of the legume lectin gene family encode proteins that function in plant defense against herbivores.

The Pto gene encodes a serine/threonine kinase that confers resistance to bacterial speck disease in tomato. Using the yeast two-hybrid system, we identified a second serine/threonine kinase, Pto-interacting 1 (Pti1), that physically interacts with Pto. Cross-phosphorylation assays revealed that Pto specifically phosphorylates Pti1 and that Pti1 does not phosphorylate Pto. Fen, another serine/threonine kinase from tomato that is closely related to Pto, was unable to phosphorylate Pti1 and was not phosphorylated by Pti1. Expression of a Pti1 transgene in tobacco plants enhanced the hypersensitive response to a P. syringae pv. tabaci strain carrying the avirulence gene avrPto. These findings indicate that Pti1 is involved in a Pto-mediated signaling pathway, probably by acting as a component downstream of Pto in a phosphorylation cascade.

A cDNA clone pCZ1, with a 1.1 kb insert, was isolated from a NaCl-adapted tobacco cell cDNA library that encodes an apparently full-length 29 kDa protein (251 amino acids) with a calculated pI of 5.7. The encoded peptide had a high amino acid sequence identity with bovine 14-3-3 protein which was originally found as an abundant protein in the animal central nervous system. Recently, proteins with sequence identity to 14-3-3 protein have also been found in plants, insects and yeast, and appear to have diverse physiological functions. Similar to the bovine brain 14-3-3 protein, the recombinant pCZ1 protein stimulated ADP-ribosylation of protein substrate by ADP-ribosyltransferase from the plant and animal pathogenic bacterium Pseudomonas aeruginosa. This recombinant protein also inhibited protein kinase C activity in vitro. Southern blot analyses indicated that most likely five genes encoding 14-3-3-like proteins are present in tobacco. The pCZ1 cDNA insert hybridized to a single mRNA of 1.1 kb from cultured tobacco cells. The level of this mRNA transcript in tobacco cells was downregulated upon adaptation to NaCl but was unaffected by short-term treatment with NaCl, ABA or ethylene. In tobacco plants, expression of transcript that hybridized to pCZ1 was tissue specific, and was most abundant in roots and flower parts. Monoclonal antibody raised against GF14 protein, a maize protein with substantial sequence identity with 14-3-3 protein detected two bands on SDS-PAGE of total proteins from unadapted tobacco cells and only a single band from cells adapted to NaCl. The GF14 antibody was also used to illustrate that the G-box element of a salt-induced gene is associated with a 14-3-3-type protein.

An Atriplex nummularia L. cDNA probe encoding the partial sequence of an isoform of the plasma-membrane H(+)-ATPase was isolated, and used to characterize the NaCl regulation of mRNA accumulation in cultured cells of this halophyte. The peptide (477 amino acids) translated from the open reading frame has the highest sequence homology to the Nicotiana plumbaginifolia plasma-membrane H(+)-ATPase isoform pma4 (greater than 80% identity) and detected a transcript of approximately 3.7 kb on Northern blots of both total and poly(A)+ RNA. The mRNA levels were comparable in unadapted cells, adapted cells (cells adapted to and growing in 342 mM NaCl) and deadapted cells (cells previously adapted to 342 mM NaCl that are now growing without salt). Increased mRNA abundance was detected in deadapted cells within 24 h after exposure to NaCl but not in unadapted cells with similar salt treatments. The NaCl up-regulation of message abundance in deadapted cells was subject to developmental control. Analogous to those reported for glycophytes, the plasma-membrane H(+)-ATPase are encoded by a multigene family in the halophyte.