Tospovirus resistance in plants

Details Tospovirus resistance in plants Tospovirus resistance in plants

1995-09-25

2001-12-11

McElwain, Elizabeth F. (Department: 1638)

Multicellular living organisms and unmodified parts thereof and

Method of introducing a polynucleotide molecule into or...

The polynucleotide confers pathogen or pest resistance

C800S301000, C536S023720, C435S418000, C435S419000

Reexamination Certificate

active

06329568

ABSTRACT:

Viruses in the Tospovirus genus infect a wide variety of plant species, particularly tobacco, peanut, vegetables and ornamental plants. Two virus species, tomato spotted wilt virus (TSWV) and impatiens necrotic spot virus (INSV) are recognized within the Tospovirus genus.
Tomato Spotted Wilt Virus (TSWV) is unique among plant viruses in that the nucleic acid-protein complex is covered by a lipoprotein envelope and it is the only thrip transmitted virus. This virus has recently been classified as the Tospovirus genus of the Bunyaviridae family. TSWV virions contain a 29K nucleocapsid protein (“NP” or “N”), two membrane-associated glycoproteins (58K and 78K) and a large 200K protein presumably for the viral transcriptase [see J. Gen. Virol. 71:2207 (1991); Virol. 56:12 (1973); and J. Gen. Virol. 36:267 (1977)]. The virus genome consists of three negative-strand (−) RNAs designated L RNA (8900 nucleotides), M RNA (5400 nucleotides) and S RNA (2900 nucleotides) [see J. Gen. Virol. 36:81 (1977); J. Gen. Virol. 53:12 (1981); and J. Gen. Virol. 70:3469 (1989)], each of which is encapsulated by the NP. The partial or full-length sequences of S RNAs from three TSWV isolates reveals the presence of two open reading frames (ORF) with an ambisense gene arrangement [see J. Gen Virol. 71:1 (1990) and J. Gen. Virol. 72:461 (1991)]. The larger open reading frame is located on the viral RNA strand and has the capacity to encode a 52K nonstructural protein. The smaller ORF is located on the viral complementary RNA strand and is translated through a subgenomic RNA into the 29K NP.
The ambisense coding strategy is also characteristic of the TSWV M RNA, with the open reading frames encoding the 58K and 78K membrane-associated glycoproteins. The TSWV L RNA has been sequenced to encode a large 200K protein presumably for the viral transcriptase.
Two TSWV serogroups, “L” and “I”, have been identified and characterized based on serological analysis of the structural proteins and morphology of cytopathic structures [see J. Gen Virol. 71:933 (1990) and Phytopathology 81:525 (1991)]. They have serologically conserved G1 and G2 glycoproteins, but the NP of the “I” serogroup is serologically distinct from that of the “L” serogroup. Comparison of the NP between the “L” and “I” serogroups has shown 62% and 67% identities at nucleotide and amino acid levels, respectively [see J. Gen. Virol. 72:2597 (1991)].
TSWV has a wide host range, infecting more than 360 plant species of 50 families and causes significant economic losses to vegetables and ornamental plants worldwide. The “L” serogroup has been found extensively in field crops such as vegetables and weeds, while the “I” serogroup has been largely confined to ornamental crops. A cucurbit isolate has recently been identified [see Plant Disease 68:1006 (1984)] as a distinct isolate because it systemically infects watermelon and other curcurbits and its NP is serologically unrelated to that of either serogroup. Although the spread of the TSWV disease can sometimes be reduced by breeding resistant plants or using non-genetic approaches, complete control of the disease by these conventional methods has generally proven to be difficult [see Plant Disease 73:375 (1989)].
Since 1986, numerous reports have shown that transgenic plants with the coat protein (CP) gene of a virus are often resistant to infection by that virus. This phenomenon is commonly referred to as coat protein-mediated protection (CPMP). The degree of protection ranges from delay in symptom expression to the absence of disease symptoms and virus accumulation. Two recent independent reports [see Biol. Technology 9:1363(1991) and Mol. Plant-Microbe Interact. 5:34 (1992)] showed that transgenic tobacco plants expressing the nucleocapsid protein (NP) gene of TSWV are resistant to infection by the homologous isolate. However, since TSWV is widespread with many biologically diverse isolates, it is very important to test the effectiveness of the transgenic plants to resist infections by different TSWV isolates. The findings of the present invention expand on those of the previous reports by demonstrating that transgenic plants according to the present invention showed resistance to two heterologous isolates of the “L” serogroup and an isolate of the “I” serogroup. We also show that resistance to the two heterologous isolates of the “L” serogroup was mainly found in plants accumulating very low, if any, levels of NP, while transgenic plants that accumulated high levels of NP were resistant to the isolate of the “I” serogroup.
However, no resistance was observed to a Brazilian isolate, although the plants that accumulated high levels of the N protein did display a delay in symptom expression. This Brazilian isolate, designated TSWV-B has the N protein that was serologically distinct from the “L” and “I” serogroups and biologically differs from a curcurbit isolate in that the TSWV-B does not systemically infect melons or squash. Therefore, one aspect of the present invention is to characterize the TSWV-B by cloning and sequencing of its S RNA and comparisons with the published sequences of other TSWV isolates.


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