A protein identification algorithm for tandem mass spectrometry by incorporating the abundance of mRNA into a binomial probability scoring model.

Peptide-spectrum matches (PSM) scoring between the experimental and theoretical spectrum is a key step in the identification of proteins using mass spectrometry (MS)-based proteomics analyses. Efficient protein identification using MS/MS data remains a challenge. The strategy of using RNA-seq data increases the number of proteins identified by re-constructing the custom search database and integrating mRNA abundance into the false discovery rate of post-PSM. However, this process lacks an algorithm that can allow the incorporation of mRNA abundance into the key scoring model of PSM. Therefore, we developed a novel PSM scoring model, which incorporates mRNA abundance for improved peptide and protein identification. In the new algorithm, abundance information of mRNA was transformed to the prior probability of protein identification and integrated to re-score in PSM using the binomial probability distribution model. Compared with other algorithms using five MS/MS datasets, the results showed that the least improvement ratios of peptide and protein groups were 3.39%-9.79% and 0.48%-8.16% in different datasets (human, rat, zebrafish, yeast, and Arabidopsis thaliana). The new strategy offers an effective solution for MS-based identification of peptides and proteins. SIGNIFICANCE: The new algorithm identifies proteins by quantifying mRNA abundance (FPKM) and incorporating it into a scoring model for peptide-spectrum matches. It is important to improve peptide and protein identification from MS/MS datasets in proteomics research.

Indole-3-acetic acid reverses the harpin-induced hypersensitive response and alters the expression of hypersensitive-response-related genes in tobacco.

Harpin proteins stimulate hypersensitive response (HR) in plants. However, the mechanism by which HR is regulated is not clear. The role of the auxin, indole-3-acetic acid (IAA), in the control of harpin-stimulated HR was investigated. IAA was used to inhibit HR that was stimulated by purified fusion harpin(Xoo) protein in tobacco. Semi-quantitative PCR and qRT-PCR were employed to detect the expression of HR related genes. IAA at 100 µM reversed harpin-induced HR which was inhibited by 500 µM 2,3,5-triiodobenzoic acid (TIBA). Semi-quantitative PCR and qRT-PCR showed the combined application of 100 µM IAA and harpin protein from Xanthomonas oryzae enhanced the expression of HR marker gene, hsr203J, but weakened the expression of the disease-defense gene, chia5. TIBA also decreased the expression of hsr203J but increased the expression of chia5. Thus, the auxin can reverse harpin(Xoo)-induced HR.

HpaXm from Xanthomonas citri subsp. malvacearum is a novel harpin with two heptads for hypersensitive response.

A novel harpin-like protein, HpaXm, was described from cotton leaf blight bacteria, Xanthomonas. citri subsp. malvacearum. The hpaXm was found to be localized between hrp2 and hrcC. A phylogenetic analysis of the complete amino acid sequence or solely the 13 highly conserved residues H2N-SEKQLDQLLTQLI-COOH in the N-terminal alpha-helix indicates that HpaXm is evolutionarily closer to HpaGXag and HpaXac than to Hpa1Xoo and Hpa1Xoc. A synthesized peptide containing two heptads, 39-LDQLLTQ-LIMALLQ-52, from the N-terminal alpha-helical region of HpaXm displayed a comparable activity in inducing HR, but other two synthesized derivatives, HpaXmDeltaT44C and HpaXmDeltaM48Q showed a reduced activity of HR. The data from a GST trap test revealed that HpaXm was released into the extracellular medium, hpaXm mutant deficient for the leader peptide (1-MNSLNTQIGANSSFL-15) was unable to be secreted outside cells but still induced HR in tobacco leaves.

Antibiotic effect of exogenously applied salicylic acid on in vitro soilborne pathogen, Fusarium oxysporum f.sp.niveum.

Salicylic acid, which is biosynthesized inside plant and is often found and accumulated in soil due to plant debris decaying, is considered as a signaling substance during plant-microbe interactions. It is involved in the cycling of biogeochemistry and related to plant resistance to biotic and abiotic stress. The antibiotic effect of salicylic acid on Fusarium oxysporum f.sp.niveum (FON) was studied to investigate the relationships between the salicylic acid and the fungus in the ecological interaction of plant-microbe. Results showed that the biomass, colony diameter, number of conidium germination and conidium production of FON were decreased by 52.0%, 25.7%, 100% and 100% at concentrations of 800 mg L(-1). However, mycotoxin yield was increased by 233%, pectinase activity raised by 168.0% and cellulase activity increased by 1325% compared to control at higher concentrations. It was concluded that salicylic acid as an allelochemical greatly inhibited FON growth and conidia formation and germination, though stimulated mycotoxin production and activities of hydrolytic enzymes by FON.

Mutations in the N-terminal coding region of the harpin protein Hpa1 from Xanthomonas oryzae cause loss of hypersensitive reaction induction in tobacco.

Harpins encoded by many gram-negative phytopathogenic bacterial hrp genes induce hypersensitive response (HR) and associated defense responses on nonhost plants. Hpa1(Xoo) and Hpa1(Xoc), two harpin proteins from Xanthomonas oryzae pathovars, induce HR when infiltrated into tobacco leaves. N- and C-terminal mutations of Hpa1(Xoo) and Hpa1(Xoc), respectively, were tested for their ability to elicit HR on tobacco. Deletion of codons for 12 highly hydrophilic amino acids (H(2)N-QGISEKQLDQLL-COOH) that partially overlap the N-terminal alpha-helical regions of respective proteins was found to be critical for the elicitation of HR in tobacco. Furthermore, two single missense mutants Hpa1(Xoo) (L51P) and Hpa1(Xoc) (L53P) that are predicted to destroy the coiled-coil integrity and inhibit the dimer formation eliminated HR elicitation activity in tobacco. However, both wild-type proteins and derivative mutants retained the ability to induce systemic acquired resistance in tobacco against tobacco mosaic virus. Accumulations of npr1 (nonexpressor of pathogenesis-related protein 1), hsr515 (hypersensitivity-related protein 515), and pr2 (pathogenesis-related protein 2) transcripts were found in tobacco plants infiltrated with wild-type or mutated proteins.