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Differential expression of in vivo and in vitro protein profile of outer membrane of Acidovorax avenae subsp. avenae.

ABSTRACT: Outer membrane (OM) proteins play a significant role in bacterial pathogenesis. In this work, we examined and compared the expression of the OM proteins of the rice pathogen Acidovorax avenae subsp. avenae strain RS-1, a Gram-negative bacterium, both in an in vitro culture medium and in vivo rice plants. Global proteomic profiling of A. avenae subsp. avenae strain RS-1 comparing in vivo and in vitro conditions revealed the differential expression of proteins affecting the survival and pathogenicity of the rice pathogen in host plants. The shotgun proteomics analysis of OM proteins resulted in the identification of 97 proteins in vitro and 62 proteins in vivo by mass spectrometry. Among these OM proteins, there is a high number of porins, TonB-dependent receptors, lipoproteins of the NodT family, ABC transporters, flagellins, and proteins of unknown function expressed under both conditions. However, the major proteins such as phospholipase and OmpA domain containing proteins were expressed in vitro, while the proteins such as the surface anchored protein F, ATP-dependent Clp protease, OmpA and MotB domain containing proteins were expressed in vivo. This may indicate that these in vivo OM proteins have roles in the pathogenicity of A. avenae subsp. avenae strain RS-1. In addition, the LC-MS/MS identification of OmpA and MotB validated the in silico prediction of the existance of Type VI secretion system core components. To the best of our knowledge, this is the first study to reveal the in vitro and in vivo protein profiles, in combination with LC-MS/MS mass spectra, in silico OM proteome and in silico genome wide analysis, of pathogenicity or plant host required proteins of a plant pathogenic bacterium.

SUBMITTER: Ibrahim M

PROVIDER: S-EPMC3499465 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Differential expression of in vivo and in vitro protein profile of outer membrane of Acidovorax avenae subsp. avenae.

Ibrahim Muhammad M Shi Yu Y Qiu Hui H Li Bin B Jabeen Amara A Li Liping L Liu He H Kube Michael M Xie Guanlin G Wang Yanli Y Blondel Carlos C Santiviago Carlos A CA Contreras Ines I Sun Guochang G

PloS one 20121115 11

Outer membrane (OM) proteins play a significant role in bacterial pathogenesis. In this work, we examined and compared the expression of the OM proteins of the rice pathogen Acidovorax avenae subsp. avenae strain RS-1, a Gram-negative bacterium, both in an in vitro culture medium and in vivo rice plants. Global proteomic profiling of A. avenae subsp. avenae strain RS-1 comparing in vivo and in vitro conditions revealed the differential expression of proteins affecting the survival and pathogenic ...[more]

PMID: 23166741

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Project description:Determining how a bacterial pathogen responds to its host and other bacterial species by altering gene expression is key to understand its pathogenesis and environmental adaption. Here, we used RNA-Seq to comprehensively and quantitatively assess the transcriptional response of the rice bacterial pathogen Acidovorax avenae subsp. avenae strain RS-1 cultivated in vitro, in vivo and in co-culture with rice rhizobacterium Burkholderia seminalis R456. Results revealed a surprisingly large number of regulatory differences between these conditions indicating adaptation of A. avenae subsp. avenae to specific ecological conditions. In particular, a number of potential virulence factors such as type 3 secretion system proteins were specifically expressed under in vivo conditions, whereas genes whose protein products are involved in inter-bacterial interaction such as auxin efflux carrier, small mechanosensitive ion channel protein, and ureidoglycolate hydrolase were among those specifically up-regulated under co-culture conditions. In addition, global genomic analysis of strain RS-1 identified 406 putative non-coding (nc) RNA genes. Interestingly, 8 ncRNA genes that were uniquely expressed under in vivo may be linked to pathogenicity while 4 ncRNA genes that were uniquely expressed under coculture conditions may be involved in adaption to co-cultivation with B. seminalis. Expression data obtained by RNA-Seq were also confirmed for selected genes by quantitative real-time PCR and two-dimensional gel electrophoresis as well as knockout analysis. Aaa strain RS-1 and B. seminalis strain R456 was isolated from diseased rice plants (Li et al., 2011; Xie et al., 2011) and rice rhizosphere (Zhang et al., 2007; Li et al., 2011), respectively, in our previous studies, and were stored in 20-30% sterile glycerol at -80°C. The samples of Aaa strain RS-1 for in vitro and in vivo analysis were prepared as described before (Ibrahim et al., 2012). The co-culture analysis of Aaa strain RS-1 with B. seminalis strain R456 were conducted according to Ruiz et al. (2009) and Di Cagno et al. (2009). Briefly, Aaa strain RS-1 and B. seminalis strain R456 was inoculated and incubated in chambers of a double culture vessel apparatus separated by a 0.4-μm membrane filter (Millipore Isopore™). In order to avoid the possible contamination during in vivo and co-culture operation, all bacterial samples were further confirmed based on the sequence analysis of 16S-rRNA (Li et al., 2011). Then samples were processed for RNA harvesting, mRNA purification and cDNA synthesis.

Project description:Sugarcane (Saccharum spp.) is an important cash crop for production of sugar and bioethanol. Red stripe caused by Acidovorax avenae subsp. avenae (Aaa) is a disease that occurs in numerous sugarcane-growing regions worldwide. In this study, 17 strains of Aaa were isolated from 13 symptomatic leaf samples in China. Nine of these strains produced white-cream colonies on nutrient agar medium while the other eight produced yellow colonies. In pairwise sequence comparisons of the 16S-23S rRNA internally transcribed spacer (ITS), the 17 strains had 98.4-100% nucleotide identity among each other and 98.2-99.5% identity with the reference strain of Aaa (ATCC 19860). Three RFLP patterns based on this ITS sequence were also found among the strains of Aaa obtained in this study. Multilocus sequence typing (MLST) based on five housekeeping genes (ugpB, pilT, lepA, trpB, and gltA) revealed that the strains of Aaa from sugarcane in China and a strain of Aaa (30179) isolated from sorghum in Brazil formed a unique evolutionary subclade. Twenty-four additional strains of Aaa from sugarcane in Argentina and from other crops worldwide were distributed in two other and separate subclades, suggesting that strains of A. avenae from sugarcane are clonal populations with local specificities. Two strains of Aaa from China (CNGX08 forming white-cream colored colonies and CNGD05 forming yellow colonies) induced severe symptoms of red stripe in sugarcane varieties LC07-150 and ZZ8 but differed based on disease incidence in two separate inoculation experiments. Infected plants also exhibited increased salicylic acid (SA) content and transcript expression of gene PR-1, indicating that the SA-mediated signal pathway is involved in the response to infection by Aaa. Consequently, red stripe of sugarcane in China is caused by genetically different strains of Aaa and at least two morphological variants. The impact of these independent variations on epidemics of red stripe remains to be investigated.

Dataset Information

Differential expression of in vivo and in vitro protein profile of outer membrane of Acidovorax avenae subsp. avenae.

Publications

Differential expression of in vivo and in vitro protein profile of outer membrane of Acidovorax avenae subsp. avenae.

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