Tetsuo Tamada

Production and pathogenicity of isolates of beet necrotic yellow vein virus with different numbers of RNA components

Summary Ten Japanese field isolates of beet necrotic yellow vein virus (BNYVV) were transmitted to Tetragonia expansa by inoculation with sap from rootlets of sugar-beet seedlings, to which the virus had been transmitted by the fungus Polymyxa betae. RNA extracted from BNYVV particles obtained from the T. expansa leaves was analysed by agarose gel electrophoresis. Some isolates contained RNA-1 (7.1 kb), RNA-2 (4.8 kb), RNA-3 (1.85 kb) and RNA-4 (1.5 kb) and the others contained, in addition, RNA-5 (1.4 kb). Further isolates, derived from single lesions produced by these isolates, had a variety of RNA compositions. Some contained only RNA-1 and RNA-2. Others contained, in addition, RNA-3, RNA-4, RNA-5 or RNA-6 (1.0 kb), or combinations of two or three of these components. Such isolates generally maintained their RNA composition on further subculture, and their particles had length distributions corresponding to their RNA components. Isolates containing RNA-1 + 2 + 3 caused yellow or strongly chlorotic local lesions in T. expansa, Beta vulgaris, B. macrocarpa and Chenopodium quinoa, and caused systemic stunting and yellow mosaic in B. macrocarpa and, occasionally, in B. vulgaris. In contrast, isolates containing RNA-1 + 2 + 4 or 1 + 2 + 5 induced chlorotic lesions, those containing RNA-1 + 2 + 6 or 1 + 2 induced faint chlorotic lesions, and none of these isolates easily infected B. macrocarpa systemically. Isolates containing different combinations of RNA-3,-4 and -5 induced more severe symptoms than those containing a single RNA. Such synergistic effects occurred between RNA-3 and RNA-4 or RNA-5, or between RNA-4 and RNA-5 or RNA-6, but not between RNA-3 and RNA-6, or between RNA-5 and RNA-6. These small RNA species therefore contain the genetic determinant(s) for lesion type and for ability to infect B. vulgaris and B. macrocarpa systemically. RNA-1 and RNA-2 are viral genome components. The other RNA components have some characteristics of viral satellite nucleic acids but they may not all be dispensable if the BNYVV isolates are to survive in nature.

Evidence that the 75K readthrough protein of beet necrotic yellow vein virus RNA-2 is essential for transmission by the fungus Polymyxa betae

Two mutant strains of beet necrotic yellow vein virus (BNYVV) containing deletion mutants of RNA-2 were produced during serial passage in mechanically inoculated Tetragonia expansa leaves. The mutant strains were referred to as S-0a (RNA-1 + 2a) and G-0b (RNA-1 + 2b). RNA-2a and RNA-2b were about 4.3 kb and 4.2 kb in length, respectively, whereas normal sized RNA-2 was about 4.8 kb in length. In vitro translation and immunoblot analysis showed that RNA-2, RNA-2a and RNA-2b all directed synthesis of the coat protein (Mr 22K). However, whereas wild-type RNA-2 also directed the synthesis of a coat protein readthrough protein with an Mr of 83K (predicted Mr 75K), RNA-2a and RNA-2b directed the production of readthrough proteins with MrS of 67K and 58K, respectively. This suggests that the deleted regions of RNA-2a and RNA-2b occur within the second open reading frame, which encodes a polypeptide of Mr 54K, which is translated by readthrough of the coat protein cistron. After the addition of wild-type RNA-3 and RNA-4 to all the strains, the mutant strains could not be transmitted by Polymyxa betae zoospores produced from either zoosporangia or resting spores, whereas the wild-type strains were readily transmitted. These results indicate that the 75K readthrough protein encoded by RNA-2 is essential for the transmission of BNYVV by P. betae.

Evidence that RNA silencing-mediated resistance to beet necrotic yellow vein virus is less effective in roots than in leaves.

In plants, RNA silencing is part of a defense mechanism against virus infection but there is little information as to whether RNA silencing-mediated resistance functions similarly in roots and leaves. We have obtained transgenic Nicotiana benthamiana plants encoding the coat protein readthrough domain open reading frame (54 kDa) of Beet necrotic yellow vein virus (BNYVV), which either showed a highly resistant or a recovery phenotype following foliar rub-inoculation with BNYVV. These phenotypes were associated with an RNA silencing mechanism. Roots of the resistant plants that were immune to foliar rub-inoculation with BNYVV could be infected by viruliferous zoospores of the vector fungus Polymyxa betae, although virus multiplication was greatly limited. In addition, virus titer was reduced in symptomless leaves of the plants showing the recovery phenotype, but it was high in roots of the same plants. Compared with leaves of silenced plants, higher levels of transgene mRNAs and lower levels of transgene-derived small interfering RNAs (siRNAs) accumulated in roots. Similarly, in nontransgenic plants inoculated with BNYVV, accumulation level of viral RNA-derived siRNAs in roots was lower than in leaves. These results indicate that the RNA silencing-mediated resistance to BNYVV is less effective in roots than in leaves.

Evidence that Beet Necrotic Yellow Vein Virus RNA-4 Is Essential for Efficient Transmission by the Fungus Polymyxa betae

Summary The efficiency of transmission by Polymyxa betae of beet necrotic yellow vein virus (BNYVV) isolates containing different RNA components was compared using sugar-beet seedlings as test plants. Isolate S-4, containing RNA-1 + 2 + 4, was transmitted by P. betae about 100 times more efficiently than isolate S-3 (RNA-1 + 2 + 3) and about 1000 times more efficiently than isolate S-0 (RNA-1 + 2). Isolate S-34 (RNA-1 + 2 + 3 + 4) was transmitted even more efficiently than isolate S-4. Each isolate retained its characteristic RNA composition after transmission by P. betae. The virus content, measured by ELISA, of infected rootlets was S-34 > S-3 > S-4 > S-0. In inoculated leaves of Tetragonia expansa and Beta macrocarpa, isolates S-3 and S-34 multiplied more extensively than did S-4 and S-0. The inefficient transmission of isolate S-3 by P. betae, as compared with S-4, cannot be attributed to a poorer ability to spread in root tissue, but the difference in transmissibility of S-3 and S-0 may be explained in this way. These results show that RNA-4 of BNYVV is essential for efficient transmission by P. betae, and suggest that RNA-3 may influence the ability of the virus to spread in root tissue. RNA-4 and RNA-3 therefore seem to play important, but different, roles in virus survival and spread in nature.

RNA 3 Deletion Mutants of Beet Necrotic Yellow Vein Virus Do Not Cause Rhizomania Disease in Sugar Beets

ABSTRACT Two mutant strains of beet necrotic yellow vein virus (BNYVV) containing deletions in RNA 3 were obtained by single lesion transfers in Tetragonia expansa. The deleted regions encode either 94 or 121 amino acids toward the C-terminal part of the 25-kDa protein (P25). Wild-type and mutant virus strains were inoculated by Polymyxa betae to sugar beet seedlings of susceptible and partially resistant cultivars. No differences were found in virus content in rootlets between mutant and wild-type viruses or between susceptible and resistant cultivars after culture for 4 weeks in a growth cabinet. However, when virus-inoculated seedlings were grown in the field for 5 months, the wild-type virus caused typical rhizomania root symptoms (69 to 96% yield loss) in susceptible cultivars, but no symptoms (23% loss) developed in most plants of the resistant cultivar, and BNYVV concentrations in the roots were 10 to 20x lower in these plants than in susceptible plants. In contrast, the mutant strains caused no symptoms in susceptible or resistant cultivars, and the virus content of roots was much lower in both cultivars than in wild-type virus infections. Wild-type RNA 3 was not detectable in most of the taproots of a resistant cultivar without any symptoms, suggesting that replication of undeleted RNA 3 was inhibited. These results indicate that the P25 of BNYVV RNA 3 is essential for the development of rhizomania symptoms in susceptible cultivars and suggest that it may fail to facilitate virus translocation from rootlets to taproots in the partially resistant cultivar.

Evidence for three groups of sequence variants of beet necrotic yellow vein virus RNA 5.

SummaryAbout half of Japanese isolates of beet necrotic yellow vein virus (BNYVV) were found to contain RNA 5 molecules, which were also detected in virus isolates from China and France. Sequence comparisons of RNA 5 (nucleotides 327 to 1171) in 25 isolates showed that there are up to 8% sequence differences, and that RNA 5 variants fall into three groups: group I contains most of the Japanese and Chinese isolates, group II two Japanese isolates, and group III four French isolates. The group I isolates fall into three small clusters. In the 26 kDa coding region of RNA 5, there was a maximum of 1.5% nucleotide sequence differences (6 amino acid changes) within the group and 8.4% nucleotide sequence differences (17 amino acid changes) between the groups. Comparisons of the coat protein gene of RNA 2 revealed that most of the Japanese and Chinese isolates belonged to the A type strain, but some isolates were of the B type. The French isolates (P type) were closely related to those of the A type. Mixed infections of the two types of virus and the two groups of RNA 5 were detected in a small area of Hokkaido. BNYVV might have been introduced into Japan and China by a similar route from at least two origins. These results, together with other evidence, suggest that the three groups of RNA 5 variants separated from an original population a long time ago and, thereafter, the group I population diverged further into three clusters, which may have been associated with the A type strain rather than the B type.

Orchid fleck virus: Brevipalpus californicus mite transmission, biological properties and genome structure.

Orchid fleck virus (OFV) causes necrotic or chlorotic ring spots and fleck symptoms in many orchid species world-wide. The virus has non-enveloped, bacilliform particles of about 40 nm × 100–150 nm and is sap-transmissible to several plant species. OFV is transmitted by the mite Brevipalpus californicus (Banks) in a persistent manner and efficiently transmitted by both adults and nymphs, but not by larvae. Viruliferous mites retain their infectivity for 3 weeks on a virus-immune host. The genome of OFV consists of two molecules of 6431 (RNA1) and 6001 nucleotides (RNA2). The RNAs have conserved and complementary terminal sequences. RNA1 contains five open reading frames (ORF), and RNA2 encodes a single ORF. Although some of the encoded proteins of OFV have sequences similar to those of proteins of plant rhabdoviruses, OFV differs from viruses in the family Rhabdoviridae in having a bipartite genome.

Identification of amino acids of the beet necrotic yellow vein virus p25 protein required for induction of the resistance response in leaves of Beta vulgaris plants

The RNA3-encoded p25 protein of beet necrotic yellow vein virus (BNYVV) is responsible for the production of rhizomania symptoms of sugar beet roots (Beta vulgaris subsp. vulgaris). Here, it was found that the presence of the p25 protein is also associated with the resistance response in rub-inoculated leaves of sugar beet and wild beet (Beta vulgaris subsp. maritima) plants. The resistance phenotype displayed a range of symptoms from no visible lesions to necrotic or greyish lesions at the inoculation site, and only very low levels of virus and viral RNA accumulated. The susceptible phenotype showed large, bright yellow lesions and developed high levels of virus accumulation. In roots after Polymyxa betae vector inoculation, however, no drastic differences in virus and viral RNA accumulation levels were found between plants with susceptible and resistant phenotypes, except at an early stage of infection. There was a genotype-specific interaction between BNYVV strains and two selected wild beet lines (MR1 and MR2) and sugar beet cultivars. Sequence analysis of natural BNYVV isolates and site-directed mutagenesis of the p25 protein revealed that 3 aa residues at positions 68, 70 and 179 are important in determining the resistance phenotype, and that host-genotype specificity is controlled by single amino acid changes at position 68. The mechanism of the occurrence of resistance-breaking BNYVV strains is discussed.

The Evolutionary History of Beet necrotic yellow vein virus Deduced from Genetic Variation, Geographical Origin and Spread, and the Breaking of Host Resistance

Beet necrotic yellow vein virus (BNYVV) is an economically important pathogen of sugar beet and has been found worldwide, probably as the result of recent worldwide spread. The BNYVV genome consists of four or five RNA components. Here, we report analysis of sequence variation in the RNA3-p25, RNA4-p31, RNA2-CP, and RNA5-p26 genes of 73 worldwide isolates. The RNA3-p25 gene encodes virulence and avirulence factors. These four sets of gene sequences each fell into two to four groups, of which the three groups of p25 formed eight subgroups with different geographical distributions. Each of these subgroup isolates (strains) could have arisen from four original BNYVV population and their mixed infections. The genetic diversity for BNYVV was relatively small. Selection pressure varied greatly depending on the BNYVV gene and geographical location. Isolates of the Italy strain, in which p25 was subject to the strongest positive selection, were able to overcome the Rz1-host resistance gene to differing degrees, whereas other geographically limited strains could not. Resistance-breaking variants were generated by p25 amino acid changes at positions 67 and 68. Our studies suggest that BNYVV originally evolved in East Asia and has recently become a pathogen of cultivated sugar beet followed by the emergence of new resistance-breaking variants.

Complete nucleotide sequence of the Japanese isolate S of beet necrotic yellow vein virus RNA and comparison with European isolates

SummaryThe complete nucleotide sequences of beet necrotic yellow vein virus RNA-1 to RNA-4 of the Japanese isolate S (BNYVV-S) were determined and compared with those of French isolate (BNYVV-F2). The nucleotide sequences of the two isolates were very similar, differing by only 1.7% (RNA-1), 4.1% (RNA-2), 2.9% (RNA-3) and 3.6% (RNA-4), respectively. The differences of the amino acid sequences of the two isolates depended upon the open reading frames (ORF) as follows: P237, 1.4%; P22 (coat protein), 2.1%; 54k ORF, 3.4%; P42, 0.5%; P13, 1.7%; P15, 3.0%; P14, 7.0% P25, 6.4%; P31, 3.5%. Comparison of the coat protein and triple gene block (P42, P13 and P15) regions of RNA-2 with other isolates revealed that BNYVV-S was much more similar to the Yugoslavian isolate (BNYVV-Yu2) than to BNYVV-F2. The nucleotide differences between BNYVV-S and BNYVV-Yu2 were less than 1%. Based upon the grouping of BNYVV variants reported by Kruse et al. [10], BNYVV-S is thus considered to belong to the A type along with BNYVV-Yu2, whereas BNYVV-F2 is classified in the B type. Our data suggest that the Japanese isolate S may have been derived from European countries other than France or Germany.