Characterization, expression, and antimicrobial activity of histones from Japanese flounder Paralichthys olivaceus.

Histone proteins are not only structurally important for chromosomal DNA packaging but also involved in the regulation of gene expression and the immune response of host against pathogens. Japanese flounder (Paralichthys olivaceus) as one of the most important marine flatfish, suffered from widespread outbreaks of diseases, and its immunological functioning remained to be elucidated. In the present study, we reported the expression patterns of four histones (H1, H2A, H3, and H3.3) and functional characterization of the histone H3.3 from flounder. Quantitative real time RT-PCR (RT-qPCR) analysis showed that expression of the four histones occurred in multiple tissues, but their levels of expression were relatively high in immune organs, and inducible in response to pathogens infection. Infection with extracellular and intracellular bacterial pathogens and viral pathogen regulated the expression of histones in a manner that depended on tissue type, pathogen, and infection stage. Specifically, H1 expression was highly induced by intracellular viral pathogens; H2AX and H3 expressions were highly induced by intracellular bacterial pathogen; dissimilarly, H3.3 expression was slightly induced by extracellular bacterial pathogen, but was inhibited by intracellular bacterial and viral pathogens. To further investigate H3.3 function, recombinant H3.3 (rH3.3) was obtained, and in vitro experiments showed rH3.3 possessed the capability of binding to both Gram-negative and Gram-positive bacteria and inhibiting the growth of some target bacteria. Consistently, In vivo results showed that overexpression of H3.3 promoted the host defense against invading pathogenic microorganism and regulated the expressions of several cytokines. These results suggested that flounder histones exhibit different expression patterns in response to the infection of different microbial pathogens, and H3.3 serves as an immune-related protein and plays an important role in antimicrobial immunity of Japanese flounder. Taken together, this study is the first report about the expression profile of different histones upon different kind of pathogens and anti-infectious immunity of H3.3 in teleost, which offered new insights into the immunological function of histones in teleost.

Major histocompatibility complex class I (MHC Iα) of Japanese flounder (Paralichthys olivaceus) plays a critical role in defense against intracellular pathogen infection.

The major histocompatibility complex (MHC) is a highly polymorphic region of the vertebrate genome that plays a critical role in initiating immune responses towards invading pathogens. It is well known that MHC I molecules play a central role in the immune response to viruses. However, rare literatures were reported the role of MHC I in the resistance to intracellular bacteria. Sequences of MHC Iα were identified in multiple teleost species, including Japanese flounder (Paralichthys olivaceus), however, the immunological function of MHC Iα remain largely unknown. In this study, we examined the expression profile and biological activity of an MHC Iα homologue, PoMHC Iα, from P. olivaceus. Structural analysis showed that PoMHC Iα possesses conserved structural characteristics of MHC Iα proteins, including MHC_I domain, IGc1 domain, transmembrane region. Expression of PoMHC Iα was upregulated in a time-dependent manner by extracellular and intracellular bacterial pathogens and viral pathogen infection. Different expression patterns were exhibited in response to the infection of different types of microbial pathogens in different immune tissues. Recombinant PoMHC Iα increased the capability of host cells to defense against intracellular pathogen Edwardsiella tarda infection and enhanced the expression of immune related genes. The knockdown of PoMHC Iα attenuated the ability of cells to eliminate E. tarda, which was sustained by the in vivo results that overexpression of PoMHC Iα promoted the host defense against invading E. tarda. Antigen uptake assay indicated PoMHC Iα participated in cells antigen presentation. Collectively, this study is the first report that MHC Iα plays an important role in immune defense against intracellular bacterial pathogen in teleost. Taken together, these findings add new insights into the biological function of teleost MHC Iα and emphasize the importance of MHC I gene products for the control of E. tarda infection.

Testifying the rice bacterial blight resistance gene xa5 by genetic complementation and further analyzing xa5 (Xa5) in comparison with its homolog TFIIAgamma1.

The recessive gene xa5 for resistance to bacterial blight resistance of rice is located on chromosome 5, and evidence based on genetic recombination has been shown to encode a small subunit of the basal transcription factor IIA (Iyer and McCouch in MPMI 17(12):1348-1354, 2004). However, xa5 has not been demonstrated by a complementation test. In this study, we introduced the dominant allele Xa5 into a homozygous xa5-line, which was developed from a cross between IRBB5 (an indica variety with xa5) and Nipponbare (a japonica variety with Xa5). Transformation of Xa5 and subsequent segregation analysis confirmed that xa5 is a V39E substitution variant of the gene for TFIIAgamma on chromosome 5 (TFIIAgamma5 or Xa5). The rice has an addition gene for TFIIAgamma exists on chromosome 1 (TFIIAgamma1). Analysis of the expression patterns of Xa5 (TFIIAgamma5)/xa5 and TFIIAgamma1 revealed that both the genes are constitutively expressed in different rice organs. However, no expression of TFIIAgamma1 could be detected in the panicle by reverse transcriptase-polymerase chain reaction. To compare the structural difference between the Xa5/xa5 and TFIIAgamma1 proteins, 3-D structures were predicted using computer-aided modeling techniques. The modeled structures of Xa5 (xa5) and TFIIAgamma1 fit well with the structure of TFIIA small subunit from human, suggesting that they may all act as a small subunit of TFIIA. The E39V substitution in the xa5 protein occurs in the alpha-helix domain, a supposed conservative substitutable site, which should not affect the basal transcription function of TFIIAgamma. The structural analysis indicates that xa5 and Xa5 potentially retain their basic transcription factor function, which, in turn, may mediate the novel pathway for bacterial blight resistance and susceptibility, respectively.

[Generation of selectable marker-free and vector backbone sequence-free Xa21 transgenic rice].

The dominant gene Xa21 with broad-spectrum and high resistance to Xanthomonas oryzae pv. oryzae (Xoo) was transferred into C418, an important restorer line of japonica hybrid rice in China using double right-border (DRB) T-DNA binary vector through Agrobacterium-mediated transformation. 17 transgenic lines were Xa21-positive with high resistance to the race P6 of Xoo through PCR analysis and resistance identification, among the total 27 independent primary transformants (T0) obtained. The subsequent analysis of the T1 progenies of these 17 T0 lines through PCR-assisted selection and resistance investigation showed that four Xa21 transgenic T0 lines could produce selectable marker-free (SMF) progenies. The frequency of primary transformants producing SMF progenies was 15%. In addition, PCR analysis also revealed these SMF progenies did not contain vector backbone sequence, and they were named as SMF and vector backbone sequence-free (SMF-VBSF) Xa21 transgenic plants. The further molecular and phenotypic analysis of the T2 and T3 progenies testified the homozygous SMF-VBSF Xa21 transgenic plants were obtained with high resistance to Xoo.