Rheumatoid arthritis (RA) is a chronic, autoimmune and inflammatory joint disease with a poorly understood etiology. Despite widespread diagnostic use of anti-citrullinated protein antibodies and rheumatoid factor proteins there is a strong demand for novel serological biomarkers to improve the diagnosis this disease. The present study was aimed to identify novel autoantigens involved in rheumatoid arthritis (RA) pathogenesis through immune-proteomic strategy. Synovial fluid samples from clinically diagnosed RA patients were separated on two-dimensional gel electrophoresis (2-DE). Samples from patients with non-RA rheumatisms (osteoarthritis and trauma) were used as controls. Immunoreactive proteins were spotted by Western blotting followed by identification through Q-TOF mass spectrometer analysis. Forty Western blots were generated using plasma from ten individual RA patients and 33 reactive spots were identified, 20 from the high molecular weight (HMW) gel and 13 from the low molecular weight (LMW) gel. Among the 33 common immunogenic spots, 18 distinct autoantigens were identified, out of which 14 are novel proteins in this context. Expression analysis of five important proteins, vimentin, gelsolin, alpha 2 HS glycoprotein (AHSG), glial fibrillary acidic protein (GFAP), and α1B-glycoprotein (A1BG) by Western blot analysis using their specific antibodies revealed their higher expression in RA synovial fluid as compared to non-RA samples. Recombinantly expressed GFAP and A1BG protein were used to develop an in-house ELISA to quantify the amount of autoantibodies in the RA patients. RA patients revealed an increase in the expression of GFAP and A1BG in the plasma as compared to osteoarthritis patients. Therefore, GFAP and A1BG can be proposed as potential new autoantigens of diagnostic importance for RA subjects. Further characterization of these proteins in rheumatoid arthritis will be helpful in understanding the role of these proteins in the disease pathogenesis providing new diagnostic tool with better specificity and accurate detection of the disease.

Heat-shock proteins (HSP) are molecular chaperones for protein molecules. These proteins play an important role in protein-protein interactions such as, folding and assisting in the establishment of proper protein conformation and prevention of unwanted protein aggregation. A small HSP gene GHSP26 present in Gossypium arboreum responds to dehydration. In the present study, an attempt was made to overcome the problem of drought stress in cotton. A cDNA of GHSP26 was isolated from G. arboreum, cloned in plant expression vector, pCAMBIA-1301 driven by the cauliflower mosaic virus 35S promoter and introduced into Gossypium hirsutum. The integration and expression studies of putative transgenic plants were performed through GUS assay; PCR from genomic DNA, and quantitative real-time PCR analysis. Transgenic cotton plants showed an enhanced drought tolerance, suggesting that GHSP26 may play a role in plant responsiveness to drought.

Two closely related genes GUSP1 and GUSP2, within the universal stress protein (USP) family, were identified and cloned from water-stressed leaves of Gossypium arboreum. GUSP1 and GUSP2 genes code for proteins with predicted molecular weights of 18.2 and 19.1 kDa, respectively. Sequence analysis showed that GUSP1 and GUSP2 are highly similar to the bacterial MJ0577-type of adenosine-triphosphate-binding Usp proteins, which have been proposed to function as a molecular switch. Nucleotide sequences of these two genes showed 81% sequence similarity while their encoded proteins share 75% amino acid homology. Both proteins have high percentages of similarity (17% to 61%) to the USPs from a variety of bacteria and plants. Real-time polymerase chain reaction expression analysis revealed a high level of GUSP gene expression in leaves, roots, and stems exclusively in plants following water stress. The highest levels of droughtinducible expression were found in the leaves. A progressive decrease in expression was observed in the stem and roots compared to very low expression in control tissues.

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The cotton (Gossypium arboreum) stress-related gene GHSP26 responds to dehydration. To elucidate its stress tolerant mechanism at the transcriptional level, we isolated and characterized the promoter region (PGHSP26, -2,831 bp) flanking the 5' GHSP26 coding region from the genomic DNA. A series of PGHSP26 deletion derivatives was created for the identification of the upstream region of the gene required for the promoter activity. Each deletion construct was analyzed by agrobacterium mediated transient transformation in tobacco leaves after treatment with abscissic acid (ABA), heavy metals and dehydration. Promoter fragments of 716 bp or longer showed two-fold or greater induction after each treatment. These findings further our understanding of the regulation of GHSP26 expression and provide a new drought-inducible promoter system in transgenic plants.

In response to water deficit stress, plants show quantitative and qualitative differences in gene expression. By using differential display and RACE (rapid amplification of cDNA ends) polymerase chain reaction (PCR) techniques an alpha crystalline‐type small heat shock protein gene (GHSP26) was isolated and characterized from Gossypium arboreum L. Alignments of 1108 bp genomic and 1026 bp cDNA sequences revealed that the GHSP26 gene comprises a single open reading frame of 230 amino acids and it contains a single intron. The gene product contains the highly conserved alpha crystalline region, spanning amino acid residues 133 to 217 and a Met‐rich region from 94 to 117aa at the N terminus. Predicted amino acid sequence shares 65%, 63%, 59%, 58%, 56%, 55%, 53%, and 22% identities with Petunia hybrida, Nicotiana tabacum, Arabidopsis thaliana, Zea mays, Agrostis stolonifera, Triticum aestivum, Oryza sative, and Nitrosococcus oceani, respectively. Expression profile of the gene was studied from leaf, stem, and root tissues, through reverse transcriptase polymerase chain reaction (RT‐PCR) and quantitative real‐time RT‐PCR analysis. The results indicated that the gene was expressed in all tissues tested in both fully hydrated and dehydrated plants. However, the gene was 100‐fold more abundant in dehydrated leaves, while only two‐fold abundant in stressed root and stem as compared to control tissues.

The evolution of the plant immune response has resulted in a highly effective defense system that is able to resist potential attack by microbial pathogens. The primary immune response is referred to as pathogen associated molecular pattern (PAMP) triggered immunity and has evolved to recognize common features of microbial pathogens. In response to the delivery of pathogen effector proteins, plants acquired R proteins to fight against pathogen attack. R-dependent defense response is important in understanding the biochemical and cellular mechanisms and underlying these interactions will enable molecular and transgenic approaches for crops with increased biotic resistance. Proteomic analyses are particularly useful for understanding the mechanisms of host plant against the pathogen attack. Recent advances in the field of proteome analyses have initiated a new research area, i.e., the analysis of more complex microbial communities and their interaction with plant. Such areas hold great potential to elucidate, not only the interactions between bacteria and their host plants, but also of bacteria-bacteria interactions between different bacterial taxa, symbiotic, pathogenic bacteria, and commensal bacteria. During biotic stress, plant hormonal signaling pathways prioritizes defense over other cellular functions. Some plant pathogens take advantage of hormone dependent regulatory system by mimicking hormones that interfere with host immune responses to promote virulence (vir). In this review, it is discussed the cross talk that plays important role in response to pathogens attack with different infection strategies using proteomic approaches.