A natural variant of a host RNA-dependent RNA polymerase is associated with increased susceptibility to viruses by Nicotiana benthamiana.

Nicotiana benthamiana often displays more intense symptoms after infection by RNA viruses than do other Nicotiana species. Here, we examined the role of RNA-dependent RNA polymerases (RdRPs) in N. benthamiana antiviral defense. cDNAs representing only two genes encoding RdRPs were identified in N. benthamiana. One RdRP was similar in sequence to SDE1/SGS2 required for maintenance of transgene silencing, whereas the second, named NbRdRP1m, was >90% identical in sequence to the salicylic acid (SA)-inducible RdRP from Nicotiana tabacum required for defense against viruses. NbRdRP1m expression was induced by SA treatment or challenge with Tobacco mosaic virus, but the gene and transcript sequences differed from those of other SA-inducible RdRPs in that they contained a 72-nt insert with tandem in-frame stop codons in the 5' portion of the ORF. N. benthamiana plants transformed with an SA-inducible RdRP gene from Medicago truncatula were more resistant to infection by Tobacco mosaic virus, Turnip vein-clearing virus, and Sunn hemp mosaic virus (members of Tobamovirus genus), but not to Cucumber mosaic virus and Potato virus X (members of different genera than the tobamoviruses). Our results indicate that N. benthamiana lacks an active SA- and virus-inducible RdRP and thus is hypersusceptible to viruses normally limited in their accumulation by this RdRP. These findings are significant for those studying virus-induced gene silencing, the hypersensitive response and systemic acquired resistance.

[1]  W. Dawson,et al.  HOST-RANGE DETERMINANTS OF PLANT VIRUSES , 1992 .

[2]  G. Macaya,et al.  Field Survey of Cucumber mosaic virus Subgroups I and II in Crop Plants in Costa Rica. , 2001, Plant disease.

[3]  Sandhya P Koushika,et al.  Loss of the Putative RNA-Directed RNA Polymerase RRF-3 Makes C. elegans Hypersensitive to RNAi , 2002, Current Biology.

[4]  Titia Sijen,et al.  On the Role of RNA Amplification in dsRNA-Triggered Gene Silencing , 2001, Cell.

[5]  J. Sherwood,et al.  Occurrence of a vein-clearing tobamovirus in turnip , 1993 .

[6]  N. Burbidge.  The Australian species of Nicotiana L. (Solanaceae). , 1960 .

[7]  H. Wheeler.  A Contribution to the Cytology of the Australian-South Pacific Species of Nicotiana. , 1945, Proceedings of the National Academy of Sciences of the United States of America.

[8]  R. S. Nelson,et al.  Mapping nucleotides in the 126-kDa protein gene that control the differential symptoms induced by two strains of tobacco mosaic virus. , 1996, Virology.

[9]  U. Melcher,et al.  Limitations to tobacco mosaic virus infection of turnip , 1999, Archives of Virology.

[10]  S. Ding,et al.  The suppressor of transgene RNA silencing encoded by Cucumber mosaic virus interferes with salicylic acid-mediated virus resistance. , 2001, Molecular plant-microbe interactions : MPMI.

[11]  K. Nishikura,et al.  A Short Primer on RNAi RNA-Directed RNA Polymerase Acts as a Key Catalyst , 2001, Cell.

[12]  G. Macino,et al.  Gene silencing in Neurospora crassa requires a protein homologous to RNA-dependent RNA polymerase , 1999, Nature.

[13]  T. Steck,et al.  RNAi in Dictyostelium: the role of RNA-directed RNA polymerases and double-stranded RNase. , 2002, Molecular biology of the cell.

[14]  D. Baulcombe,et al.  Gene Silencing without DNA: RNA-Mediated Cross-Protection between Viruses , 1999, Plant Cell.

[15]  J. Carrington,et al.  Cap-independent enhancement of translation by a plant potyvirus 5' nontranslated region , 1990, Journal of virology.

[16]  R. Beachy,et al.  Influence of heterologous tobamovirus movement protein and chimeric-movement protein genes on cell-to-cell and long-distance movement. , 1994, Virology.

[17]  Ming-Bo Wang,et al.  Gene silencing as an adaptive defence against viruses , 2001, Nature.

[18]  H. Sambrook.  Molecular cloning : a laboratory manual. Cold Spring Harbor, NY , 1989 .

[19]  P. Dijk,et al.  Accessions of Australian Nicotiana species suitable as indicator hosts in the diagnosis of plant virus diseases , 1987, Netherlands Journal of Plant Pathology.

[20]  Tamas Dalmay,et al.  An RNA-Dependent RNA Polymerase Gene in Arabidopsis Is Required for Posttranscriptional Gene Silencing Mediated by a Transgene but Not by a Virus , 2000, Cell.

[21]  D. Bamford,et al.  Cellular RNA-dependent RNA polymerase involved in posttranscriptional gene silencing has two distinct activity modes. , 2002, Molecular cell.

[22]  Diqiu Yu,et al.  Analysis of the involvement of an inducible Arabidopsis RNA-dependent RNA polymerase in antiviral defense. , 2003, Molecular plant-microbe interactions : MPMI.

[23]  Jill M. Spoerke,et al.  EGO-1 is related to RNA-directed RNA polymerase and functions in germ-line development and RNA interference in C. elegans , 2000, Current Biology.

[24]  H. L. Sänger,et al.  Isolation of an RNA-Directed RNA Polymerase–Specific cDNA Clone from Tomato , 1998, Plant Cell.

[25]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[26]  R. Plasterk,et al.  The genetics of RNA silencing. , 2002, Annual review of genetics.

[27]  J. O. Berry,et al.  Salicylic Acid Can Induce Resistance to Plant Virus Movement , 1998 .

[28]  R. S. Nelson,et al.  Accumulation of mild and severe strains of tobacco mosaic virus in minor veins of tobacco , 1995 .

[29]  J. Carr,et al.  Salicylic Acid Has Cell-Specific Effects on Tobacco mosaic virus Replication and Cell-to-Cell Movement1 , 2002, Plant Physiology.

[30]  J. Carr,et al.  Salicylic Acid Interferes with Tobacco Mosaic Virus Replication via a Novel Salicylhydroxamic Acid-Sensitive Mechanism. , 1997, The Plant Cell.

[31]  M. Kumagai,et al.  Cytoplasmic inhibition of carotenoid biosynthesis with virus-derived RNA. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[32]  J. Carr,et al.  Genetic Modification of Alternative Respiration Has Differential Effects on Antimycin A-Induced versus Salicylic Acid-Induced Resistance to Tobacco mosaic virus1 , 2003, Plant Physiology.

[33]  R. S. Nelson,et al.  Vascular invasion routes and systemic accumulation patterns of tobacco mosaic virus in Nicotiana benthamiana. , 2000, The Plant journal : for cell and molecular biology.

[34]  Olivier Voinnet,et al.  Initiation and Maintenance of Virus-Induced Gene Silencing , 1998, Plant Cell.

[35]  B. Fan,et al.  An important role of an inducible RNA-dependent RNA polymerase in plant antiviral defense , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[36]  D. Baulcombe,et al.  Jellyfish green fluorescent protein as a reporter for virus infections. , 1995, The Plant journal : for cell and molecular biology.

[37]  R. Schilperoort,et al.  Plant Molecular Biology Manual , 1989, Springer Netherlands.

[38]  Phillip D Zamore,et al.  Ancient Pathways Programmed by Small RNAs , 2002, Science.

[39]  C. Shewmaker,et al.  Structure and expression of elongation factor 1α in tomato , 1989 .

[40]  Jennifer W. Weller,et al.  The Medicago Genome Initiative: a model legume database , 2001, Nucleic Acids Res..

[41]  Philippe Mourrain,et al.  Arabidopsis SGS2 and SGS3 Genes Are Required for Posttranscriptional Gene Silencing and Natural Virus Resistance , 2000, Cell.

[42]  M. Ravelonandro,et al.  Molecular studies of the synergistic interactions between Plum pox virus HC-Pro protein and Potato virus X , 2002, Archives of Virology.