The coat protein of potato virus X is a strain-specific elicitor of Rx1-mediated virus resistance in potato

The Rx1 gene in potato confers extreme resistance to potato virus X (PVX). To investigate the mechanism and elicitation of Rx resistance, protoplasts of potato cv. Cara (Rx1 genotype) and Maris Bard (rx1 genotype) were inoculated with PVX and tobacco mosaic virus (TMV). At 24 h post-inoculation in Maris Bard protoplasts there was at least 100-fold more PVX RNA than in protoplasts of Cara. TMV RNA accumulated to the same level in both types of protoplast. However, when the TMV was inoculated together with PVX the accumulation of TMV RNA was suppressed in the Cara (Rx1 genotype) protoplasts to the same extent as PVX. The Rx1 resistance also suppressed accumulation of a recombinant TMV in which the coat protein gene was replaced with the coat protein gene of PVX. It is therefore concluded that Rx1-mediated resistance is elicited by the PVX coat protein, independently of any other proteins encoded by PVX. The domain of the coat protein with elicitor activity was localized by deletion and mutation analysis to the structural core of a non-virion form of the coat protein.     

Processing of Bacillus subtilis small cytoplasmic RNA: evidence for an additional endonuclease cleavage site

Small cytoplasmic RNA (scRNA) of Bacillus subtilis is the RNA component of the signal recognition particle. scRNA is transcribed as a 354-nt precursor, which is processed to the mature 271-nt scRNA. Previous work demonstrated the involvement of the RNase III-like endoribonuclease, Bs-RNase III, in scRNA processing. Bs-RNase III was found to cleave precursor scRNA at two sites (the 5′ and 3′ cleavage sites) located on opposite sides of the stem of a large stem-loop structure, yielding a 275-nt RNA, which was then trimmed by a 3′ exoribonuclease to the mature scRNA. Here we show that Bs-RNase III cleaves primarily at the 5′ cleavage site and inefficiently at the 3′ site. RNase J1 is responsible for much of the cleavage that releases scRNA from downstream sequences. The subsequent exonucleolytic processing is carried out largely by RNase PH.     

TGBp3 triggers the unfolded protein response and SKP1‐dependent programmed cell death

The Potato virus X (PVX) triple gene block protein 3 (TGBp3), an 8‐kDa membrane binding protein, aids virus movement and induces the unfolded protein response (UPR) during PVX infection. TGBp3 was expressed from the Tobacco mosaic virus (TMV) genome (TMV‐p3), and we noted the up‐regulation of SKP1 and several endoplasmic reticulum (ER)‐resident chaperones, including the ER luminal binding protein (BiP), protein disulphide isomerase (PDI), calreticulin (CRT) and calmodulin (CAM). Local lesions were seen on leaves inoculated with TMV‐p3, but not TMV or PVX. Such lesions were the result of TGBp3‐elicited programmed cell death (PCD), as shown by an increase in reactive oxygen species, DNA fragmentation and induction of SKP1 expression. UPR‐related gene expression occurred within 8 h of TMV‐p3 inoculation and declined before the onset of PCD. TGBp3‐mediated cell death was suppressed in plants that overexpressed BiP, indicating that UPR induction by TGBp3 is a pro‐survival mechanism. Anti‐apoptotic genes Bcl‐xl, CED‐9 and Op‐IAP were expressed in transgenic plants and suppressed N gene‐mediated resistance to TMV, but failed to alleviate TGBp3‐induced PCD. However, TGBp3‐mediated cell death was reduced in SKP1‐silenced Nicotiana benthamiana plants. The combined data suggest that TGBp3 triggers the UPR and elicits PCD in plants.     

A dual gene‐silencing vector system for monocot and dicot plants

Plant virus‐based gene‐silencing vectors have been extensively and successfully used to elucidate functional genomics in plants. However, only limited virus‐induced gene‐silencing (VIGS) vectors can be used in both monocot and dicot plants. Here, we established a dual gene‐silencing vector system based on Bamboo mosaic virus (BaMV) and its satellite RNA (satBaMV). Both BaMV and satBaMV vectors could effectively silence endogenous genes in Nicotiana benthamiana and Brachypodium distachyon. The satBaMV vector could also silence the green fluorescent protein (GFP) transgene in GFP transgenic N. benthamiana. GFP transgenic plants co‐agro‐inoculated with BaMV and satBaMV vectors carrying sulphur and GFP genes, respectively, could simultaneously silence both genes. Moreover, the silenced plants could still survive with the silencing of genes essential for plant development such as heat‐shock protein 90 (Hsp90) and Hsp70. In addition, the satBaMV‐ but not BaMV‐based vector could enhance gene‐silencing efficiency in newly emerging leaves of N. benthamiana deficient in RNA‐dependant RNA polymerase 6. The dual gene‐silencing vector system of BaMV and satBaMV provides a novel tool for comparative functional studies in monocot and dicot plants.     

A chimeric Potato virus X encoding a heterologous peptide affects Nicotiana benthamiana chloroplast structure

Abstract

The cytopathology of a Potato virus X (PVX) recombinant variant (encoding as fusion of an epitope of immunological interest with the N‐terminus of the coat protein, PVXSmaP18DD) has been compared with that induced by the wild‐type virus (PVX wt) in Nicotiana benthamiana plants. Both PVX wt and PVXSmaP18DD caused similar ultrastructural alterations, characterized by the presence of laminated inclusion components and bulk virus accumulations in mesophyll cells. However, some striking differences were observed not only in the morphology of these accumulations (typically ordered in PVX wt infection and disordered in PVXSmaP18DD infection) but also because the chimeric virus caused peculiar alterations in chloroplasts structure.

Abbreviations: CP, coat protein; d.p.i., days post inoculation; LIC, laminated inclusion components; PVX, Potato virus X     

Influence of cytoplasmic heat shock protein 70 on viral infection of Nicotiana benthamiana

The accumulation of heat shock protein 70 (Hsp70) generally occurs in plants infected with viruses. However, the effect of Hsp70 accumulation on plant viral infection and pathogenesis remains elusive. In this study, the expression of six Hsp70 genes was found to be induced by the four diverse RNA viruses, Tobacco mosaic virus, Potato virus X (PVX), Cucumber mosaic virus and Watermelon mosaic virus, in Nicotiana benthamiana. Heat treatment enhanced the accumulation and systemic infection of these viruses. Similar results were obtained for viral infection in plants heterologously expressing an Arabidopsis cytoplasmic Hsp70 through either a PVX vector or Agrobacterium infiltration. In contrast, viral infection was compromised in cytoplasmic NbHsp70c‐1 gene‐silenced plants. These data demonstrate that the cytoplasmic Hsp70s can enhance the infection of N. benthamiana by diverse viruses.     

Disruption of the methionine cycle and reduced cellular gluthathione levels underlie potex–potyvirus synergism in Nicotiana benthamiana

Infection caused by the synergistic interaction of two plant viruses is typically manifested by severe symptoms and increased accumulation of either virus. In potex–potyviral synergism, the potyviral RNA silencing suppressor helper component proteinase (HCPro) is known to enhance the pathogenicity of the potexvirus counterpart. In line with this, Potato virus X (PVX; genus Potexvirus) genomic RNA (gRNA) accumulation and gene expression from subgenomic RNA (sgRNA) are increased in Nicotiana benthamiana by Potato virus A (PVA; genus Potyvirus) HCPro expression. Recently, we have demonstrated that PVA HCPro interferes with the host cell methionine cycle by interacting with its key enzymes S‐adenosyl‐l ‐methionine synthetase (SAMS) and S‐adenosyl‐l ‐homocysteine hydrolase (SAHH). To study the involvement of methionine cycle enzymes in PVX infection, we knocked down SAMS and SAHH. Increased PVX sgRNA expression between 3 and 9 days post‐infiltration (dpi) and upregulation of (–)‐strand gRNA accumulation at 9 dpi were observed in the SAHH‐silenced background. We found that SAMS and SAHH silencing also caused a significant reduction in glutathione (GSH) concentration, specifically in PVX‐infected plants between 2 and 9 dpi. Interestingly, HCPro expression in PVX‐infected plants caused an even stronger reduction in GSH levels than did SAMS + SAHH silencing and a similar level of reduction was also achieved by knocking down GSH synthetase. PVX sgRNA expression was increased in the GSH synthetase‐silenced background. GSH is a major antioxidant of plant cells and therefore GSH shortage may explain the strong oxidative stress and severe symptoms observed during potex–potyvirus mixed infection.     

Observation of Potato Virus X in Tobacco Mosaic Virus (TMV)- Induced Local Lesions and Surrounding Zones in Leaves of Datura stramonium L. in Mixed Infection

In mixed infection by Tobacco Mosaic Virus (TMV) and Potato Virus X (PVX) of leaves of Datura stramonium L., PVX particles were observed in the developing local lesions in both the central part and on the periphery, in addition to TMV. PVX virions were found either separately or together with TMV. Sometimes in local lesions mainly in their periphery, PVX-specific laminar inclusion components were observed and, in certain cases, cylindrical bodies about 120—140 nm in diameter. In 2 mm surrounding zones from the edge of the lesions, TMV particles were not observed. However, in the majority of cells of these zones, PVX intensively accumulated, often forming large masses. In some cases, we observed parts of cells with relatively small amounts of dispersed PVX particles, associated with laminar inclusion bodies. In cell areas with large accumulations of PVX, laminar inclusions were not found.     

Identification of tobacco proteins associated with the stem-loop 1 RNAs of <Emphasis Type="Italic">Potato virus X</Emphasis>

Potato virus X (PVX) contains five viral proteins as well as cis-acting elements like stem-loop 1 (SL1) RNAs at the 5′ region. SL1 RNAs are involved in PVX RNA replication, encapsidation, translation, and cell-to-cell movement. In this study, we performed two-dimensional electrophoresis Northwestern blot analysis and matrix-assisted laser desorption ionization time of flight mass spectrometry and identified 24 tobacco proteins that interact with SL1 RNAs. Interestingly, one-third of the identified host proteins have been shown to interact with many plant viral proteins. In addition, we demonstrated that PVX capsid protein can bind to both SL1(+/−) RNAs. We further selected three Nicotiana benthamiana proteins including NbMPB2Cb, NbMBF1, and NbCPIP2a, to confirm results of Northwestern blot analysis. Electrophoretic mobility shift assay showed that NbMPB2Cb and NbMBF1 bind to both SL1(+/−) RNAs in vitro. In contrast, NbCPIP2a interacts only SL1(+) RNA. Taken together, we provide a list of host proteins interacting with PVX SL1 RNAs, which would be good candidates for the study of viral RNA-host protein interaction.     

地中海富盐菌中非编码RNA的鉴定与分析

【目的】通过转录组高通量测序技术(即RNA-seq),结合生物信息学分析和分子生物学方法,在组学水平鉴定极端嗜盐菌中可能的非编码RNA(nc RNA)。【方法】将培养至对数中期的地中海富盐菌在不同盐浓度下处理30分钟,提取RNA,进行链特异的转录组测序和5′端区分的转录组测序,通过生物信息学分析在全基因组范围内鉴定nc RNA,预测其转录边界;然后通过Northern blot和环化RNA反转录聚合酶链式反应(CR-RT-PCR)对部分预测的nc RNA进行实验验证。【结果】比较两种RNA-seq技术在不同培养条件下的RNA测序结果和对转录单元的精细分析,共鉴定到105个高可信度的nc RNA,并发现4个在不同盐度下表达差异较大的nc RNA,通过Northern blot和CR-RT-PCR验证了inc RNA1436和inc RNA1903的表达情况、转录本、转录起始位点及终止位点等。【结论】首次在组学水平鉴定了地中海富盐菌中的nc RNA,不同盐浓度刺激下部分nc RNA的转录差异暗示其有可能参与地中海富盐菌对盐胁迫的适应,高可信度nc RNA的组学发现为今后全面开展嗜盐古菌nc RNA的功能机制研究提供了基础数据及重要的切入点。