Screening and analysis of genes expressed upon infection of broad bean with Clover yellow vein virus causing lethal necrosis
© Nakahara et al; licensee BioMed Central Ltd. 2011
Received: 16 February 2011
Accepted: 18 July 2011
Published: 18 July 2011
Clover yellow vein virus (ClYVV) causes lethal systemic necrosis in legumes, including broad bean (Vicia faba) and pea (Pisum sativum). To identify host genes involved in necrotic symptom expression after ClYVV infection, we screened cDNA fragments in which expression was changed in advance of necrotic symptom expression in broad bean (V. faba cv. Wase) using the differential display technique and secondarily with Northern blot analysis. Expression changes were confirmed in 20 genes, and the six that exhibited the most change were analyzed further. These six genes included a gene that encodes a putative nitrate-induced NOI protein (VfNOI), and another was homologous to an Arabidopsis gene that encodes a glycine- and proline-rich protein GPRP (VfGPRP). We recently reported that necrotic symptom development in ClYVV-infected pea is associated with expression of salicylic acid (SA)-dependent pathogenesis-related (PR) proteins and requires SA-dependent host responses. Interestingly, VfNOI and VfGPRP expression was correlated with that of the putative SA-dependent PR proteins in ClYVV-infected broad bean. However, broad bean infected with a recombinant ClYVV expressing the VfGPRP protein showed weaker symptoms and less viral multiplication than that infected with ClYVV expressing the GFP protein. These results imply that VfGPRP plays a role in defense against ClYVV rather than in necrotic symptom expression.
Virus infections induce a number of disease symptoms such as developmental abnormalities, chlorosis, and necrosis in plants. These symptom expressions not only result from disruption of cell activity by virus infection but also from the complex interplay between plant and virus. Although it is broadly true that symptom severity, e.g., latent to lethal, depends on both viral pathogenicity and plant susceptibility, elucidating how these symptoms are expressed is an enormous challenge to plant virologists.
Recent studies regarding expression analysis of a large number of genes with microarrays have shown that sets of defense-related genes are expressed upon infection of a susceptible plant with several different viruses [1, 2], suggesting that even susceptible plants recognize virus infection and mount defense responses. Ironically, in several plant-virus pathosystems, close relationships between defense responses to virus infection and severe necrotic symptoms have been reported [3–7]; these defense responses are thought to involve a hypersensitive response (HR) and programmed cell death (PCD), which are mediated by recognition of virus infection through a resistance (R) gene, which encodes a nucleotide-binding-leucine-rich repeat (NB-LRR) protein that detects a specific viral avirulence (Avr) gene. Although the way in which these defense responses are associated with necrotic symptoms largely remains to be examined, one likely explanation is that when a plant bearing an R gene is infected with a virus possessing a corresponding Avr gene and fails to control the spread of the virus throughout the entire plant, HR-PCD would necessarily occur wherever the virus infects, resulting in systemic necrosis [4, 8]. We have recently shown that the lethal systemic necrosis observed in legumes infected with Clover yellow vein virus (ClYVV) is one such case involving defense responses [9, 10]. Therefore, it is hardly surprising that the screening of host genes whose expression is elicited by ClYVV infection identifies genes involved in defense responses related to HR-PCD. The objective of the screening is, however, aimed at understanding the molecular mechanism of necrotic symptom expression in legumes infected with ClYVV.
To identify host genes whose expression changes are not just the outcome of necrotic symptoms but are a cause of the symptoms, gene expression was analyzed in broad bean before the development of ClYVV necrotic symptoms. Total RNAs were extracted from non-inoculated upper leaves of ClYVV and mock-inoculated plants at 1, 2, and 4 dpi with Trizol reagent (Invitrogen, Carlsbad, CA, USA), basically in accordance with the manufacturer's instructions (Figure 1), and their cDNA populations were compared using differential display. Fluorescent cDNAs were generated from the extracted total RNA by reverse transcription (RT) coupled with polymerase chain reaction (PCR) using an Enzyme Set-FDD and Fluorescence Differential Display Kit (TaKaRa, Shiga, Japan) according to the manufacturer's instructions. PCR products amplified using 96 different binary combinations with four fluorescent downstream primers and 24 upstream primers were fractionated in electrophoresis on 6% Long Ranger acrylamide gel (BioWhittaker Molecular Applications, Rockland, ME, USA) containing 8 M Urea. The fluorescent amplified cDNA fragments were visualized using an FMBIO II image analyzer (TaKaRa, Ohtsu, Japan).
The six cDNA fragments whose expressions changed upon infection of broad bean with ClYVV
Flagments (deduced amino acid sequence)1
Homologous proteins (amino acid sequence)
k47 (199 aa)
Glutathione S-transferase GST 16 protein (221 aa)
k58 (79 aa)
Putative NOI protein, nitrate-induced protein (79 aa)
k158 (202 aa)
GPRP (177 aa)
AtGPRP2 (179 aa)
m2 (112 aa)
Putative senescence-associated protein (279 aa)
m4 (74 aa)
Putative thaumatin-like protein (224 aa)
m18 (34 aa)
Quinone reductase family protein (205 aa)
The correlation of k58 and k158 expression with necrosis was further examined in a different host, pea (Pisum sativum). When pea line PI 343958 expressed necrotic spots under either ClYVV or ClYVV/CN-NdB infection [9, 10], k58 and k158 expression, as well as expression of aCHI and hsr203J, was also elicited by either infection (Figure 2D). Nevertheless, k58 and k158 were not elicited in the same pea line (PI 343958) inoculated with Bean yellow mosaic virus (BYMV)-CS , which is another potyvirus closely related to ClYVV in terms of genome nucleotide sequences and host range, and does not cause necrosis, but does result in mosaic symptoms in pea (Figure 2E). Taken together, k58 and k158 expression was constantly correlated with aCHI, hsr203J, and necrotic symptom expression in legumes infected with ClYVV, raising the possibility of their involvement in the SA-dependent defense responses required for necrotic symptom expression.
We thank C. Masuta for technical advice. This work was supported by JSPS/MEXT KAKENHI (17780032, 18108001, 20688002).
- Whitham SA, Quan S, Chang HS, Cooper B, Estes B, Zhu T, Wang X, Hou YM: Diverse RNA viruses elicit the expression of common sets of genes in susceptible Arabidopsis thaliana plants. Plant J 2003, 33: 271-283. 10.1046/j.1365-313X.2003.01625.xView ArticlePubMedGoogle Scholar
- Whitham SA, Yang C, Goodin MM: Global impact: elucidating plant responses to viral infection. Mol Plant Microbe Interact 2006, 19: 1207-1215. 10.1094/MPMI-19-1207View ArticlePubMedGoogle Scholar
- Komatsu K, Hashimoto M, Ozeki J, Yamaji Y, Maejima K, Senshu H, Himeno M, Okano Y, Kagiwada S, Namba S: Viral-induced systemic necrosis in plants involves both programmed cell death and the inhibition of viral multiplication, which are regulated by independent pathways. Mol Plant Microbe Interact 2010, 23: 283-293. 10.1094/MPMI-23-3-0283View ArticlePubMedGoogle Scholar
- Dinesh-Kumar SP, Tham WH, Baker BJ: Structure-function analysis of the tobacco mosaic virus resistance gene N . Proc Natl Acad Sci USA 2000, 97: 14789-14794. 10.1073/pnas.97.26.14789PubMed CentralView ArticlePubMedGoogle Scholar
- Hajimorad MR, Eggenberger AL, Hill JH: Loss and gain of elicitor function of Soybean mosaic virus G7 provoking Rsv1 -mediated lethal systemic hypersensitive response maps to P3. J Virol 2005, 79: 1215-1222. 10.1128/JVI.79.2.1215-1222.2005PubMed CentralView ArticlePubMedGoogle Scholar
- Fisher ML, Kyle MM: Inheritance of resistance to potyviruses in Phaseolus-vulgaris L.3. Cosegregation of phenotypically similar dominant responses to 9 potyviruses. Theor Appl Genet 1994, 89: 818-823.PubMedGoogle Scholar
- Kim B, Masuta C, Matsuura H, Takahashi H, Inukai T: Veinal necrosis induced by turnip mosaic virus infection in Arabidopsis is a form of defense response accompanying HR-like cell death. Mol Plant Microbe Interact 2008, 21: 260-268. 10.1094/MPMI-21-2-0260View ArticlePubMedGoogle Scholar
- Collmer CW, Marston MF, Taylor JC, Jahn M: The I gene of bean: a dosage-dependent allele conferring extreme resistance, hypersensitive resistance, or spreading vascular necrosis in response to the potyvirus Bean common mosaic virus . Mol Plant Microbe Interact 2000, 13: 1266-1270. 10.1094/MPMI.2000.13.11.1266View ArticlePubMedGoogle Scholar
- Atsumi G, Kagaya U, Kitazawa H, Nakahara KS, Uyeda I: Activation of the salicylic acid signaling pathway enhances Clover yellow vein virus virulence in susceptible pea cultivars. Mol Plant Microbe Interact 2009, 22: 166-175. 10.1094/MPMI-22-2-0166View ArticlePubMedGoogle Scholar
- Ravelo G, Kagaya U, Inukai T, Sato M, Uyeda I: Genetic analysis of lethal tip necrosis induced by Clover yellow vein virus infection in pea. J Gen Plant Pathol 2007, 73: 59-65. 10.1007/s10327-006-0324-6View ArticleGoogle Scholar
- Masuta C, Yamana T, Tacahashi Y, Uyeda I, Sato M, Ueda S, Matsumura T: Development of clover yellow vein virus as an efficient, stable gene-expression system for legume species. Plant J 2000, 23: 539-546. 10.1046/j.1365-313x.2000.00795.xView ArticlePubMedGoogle Scholar
- Yambao ML, Yagihashi H, Sekiguchi H, Sekiguchi T, Sasaki T, Sato M, Atsumi G, Tacahashi Y, Nakahara KS, Uyeda I: Point mutations in helper component protease of clover yellow vein virus are associated with the attenuation of RNA-silencing suppression activity and symptom expression in broad bean. Arch Virol 2008, 153: 105-115. 10.1007/s00705-007-1073-3View ArticlePubMedGoogle Scholar
- Kume H, Tanaka S, Murayama D: A strain of bean yellow mosaic virus isolated from mosaic-diseased red clover plant. Mem Fac Agr Hokkaido Univ 1970, 7: 435-449.Google Scholar
- Marty I, Monfort A, Stiefel V, Ludevid D, Delseny M, Puigdomenech P: Molecular characterization of the gene coding for GPRP, a class of proteins rich in glycine and proline interacting with membranes in Arabidopsis thaliana . Plant Mol Biol 1996, 30: 625-636. 10.1007/BF00049336View ArticlePubMedGoogle Scholar
- Sato M, Masuta C, Uyeda I: Natural resistance to Clover yellow vein virus in beans controlled by a single recessive locus. Mol Plant Microbe Interact 2003, 16: 994-1002. 10.1094/MPMI.2003.16.11.994View ArticlePubMedGoogle Scholar
- Neale AD, Blomstedt CK, Bronson P, Le TN, Guthridge K, Evans J, Gaff DF, Hamill JD: The isolation of genes from the resurrection grass Sporobolus stapfianus which are induced during severe drought stress. Plant Cell Environ 2000, 23: 265-277. 10.1046/j.1365-3040.2000.00548.xView ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.