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Detection and characterization of spontaneous internal deletion mutants of Beet Necrotic yellow vein virus RNA3 from systemic host Nicotiana benthamiana.

Wang Y, Fan H, Wang XB, Li M, Han C, Li D, Yu J - Virol. J. (2011)

Bottom Line: Our studies demonstrated the internal deletion mutants of BNYVV-RNA3 were spontaneously generated in the systemic infection on N. benthamiana.The internal deletions didn't affect the efficient replication of D-RNA3s, instead by improving the stability and pathogenicity of RNA3 in the systemic host N. benthamiana.Besides, our results also suggested the downstream N protein of RNA3, but not the upstream p25 protein, may play an important role in the systemic infection on N. benthamiana.

View Article: PubMed Central - HTML - PubMed

Affiliation: State Key Laboratory for Agro-biotechnology and Ministry of Agriculture Key Laboratory for Plant Pathology, China Agricultural University, Beijing 100193, PR China.

ABSTRACT

Background: Beet Necrotic Yellow Vein virus (BNYVV) is a member of the genus Benyvirus causing a worldwide sugar beet disease rhizomania. BNYVV contains four or five plus-sense single stranded RNAs. In altered selective conditions, multipartite RNA viruses of plant are prone to undergoing internal deletions, thus turning into Defective RNAs (D RNAs). Although several D RNAs have been reported in BNYVV infection, the spontaneous internal deletion mutants responsible for severe symptom in systemic host Nicotiana benthamiana (N. benthamiana) are not described so far.

Results: Systemic host N. benthamiana was inoculated by Chinese BNYVV isolates. RT-PCR and Northern blot showed that the D RNAs forms of BNYVV RNA3 were present in the systemic infection of the N. benthamiana. Three distinct D-RNA3s, named as D-RNA 3α, D-RNA 3β and D-RNA 3γ, were made into infectious clones. When inoculated on the N. benthamiana, the in vitro transcripts of D forms exhibited more stable than that of wild-type RNA3 in systemic movement. Among the detected mutant, the p25 protein frame-shift mutant (D-RNA3α) induced obvious necrotic lesions on Tetragonia.expansa (T. expansa) and pronounced systemic symptom on the N. benthamiana. The D-RNA3α was further mutated artificially to pre-terminate the downstream N protein, leading to the abolishment of the pathogenicity, indicating the N protein was responsible for the necrotic symptom.

Conclusion: Our studies demonstrated the internal deletion mutants of BNYVV-RNA3 were spontaneously generated in the systemic infection on N. benthamiana. The internal deletions didn't affect the efficient replication of D-RNA3s, instead by improving the stability and pathogenicity of RNA3 in the systemic host N. benthamiana. Besides, our results also suggested the downstream N protein of RNA3, but not the upstream p25 protein, may play an important role in the systemic infection on N. benthamiana.

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Effects of BNYVV RNA3 and D-RNA3 mutants on the symptom of local lesion host. (A) Symptom induced by RNA1, 2, alone or combined with in vitro transcripts of RNA3 and D-RNA3 on the inoculated leaves of T.expansa in 5 dpi. (B) Northern analysis of wild type RNA3 and D-RNA3 mutants from inoculated leaves of T.expansa. (C) Symptom induced by RNA1 2, alone or combined with in vitro transcripts of RNA3 and D-RNA3α on the inoculated leaves of C. amaranticolor in 5 dpi.
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Figure 3: Effects of BNYVV RNA3 and D-RNA3 mutants on the symptom of local lesion host. (A) Symptom induced by RNA1, 2, alone or combined with in vitro transcripts of RNA3 and D-RNA3 on the inoculated leaves of T.expansa in 5 dpi. (B) Northern analysis of wild type RNA3 and D-RNA3 mutants from inoculated leaves of T.expansa. (C) Symptom induced by RNA1 2, alone or combined with in vitro transcripts of RNA3 and D-RNA3α on the inoculated leaves of C. amaranticolor in 5 dpi.

Mentions: To further investigate the function of D-RNA3s, the full-length cDNA clones of the three D-RNA3s were constructed and inoculated on the local host. The classical local host T. expansa of BNYVV could be induced to form faint chlorotic spots by RNA1 and 2 (isolate BN12, Figure 3A). In contrast, the co-infection of full-length RNA3 and BN12 would cause yellow spots in 5 dpi, as described previously (Figure 3A) [17]. The D-RNA 3β or D-RNA 3γ, as did BN12 alone, only caused faint chlorotic spots, demonstrating the deletion form of p25 protein was deficient for inducing yellow spots. Surprisingly, the D-RNA3α induced obvious necrotic spots on T. expansa in 3 dpi, and then some yellow halos were shown around the necrotic spot in the few days later (Figure 3A). In the C. amaranticolor, the D-RNA3α could also induce necrotic spot in the inoculated leaves (Figure 3C). The northern results showed the D-RNA3s form could be replicated as efficiently as wild type RNA3, indicating the deletion region had no effect on the replication of RNA3 (Figure 3B).


Detection and characterization of spontaneous internal deletion mutants of Beet Necrotic yellow vein virus RNA3 from systemic host Nicotiana benthamiana.

Wang Y, Fan H, Wang XB, Li M, Han C, Li D, Yu J - Virol. J. (2011)

Effects of BNYVV RNA3 and D-RNA3 mutants on the symptom of local lesion host. (A) Symptom induced by RNA1, 2, alone or combined with in vitro transcripts of RNA3 and D-RNA3 on the inoculated leaves of T.expansa in 5 dpi. (B) Northern analysis of wild type RNA3 and D-RNA3 mutants from inoculated leaves of T.expansa. (C) Symptom induced by RNA1 2, alone or combined with in vitro transcripts of RNA3 and D-RNA3α on the inoculated leaves of C. amaranticolor in 5 dpi.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3142242&req=5

Figure 3: Effects of BNYVV RNA3 and D-RNA3 mutants on the symptom of local lesion host. (A) Symptom induced by RNA1, 2, alone or combined with in vitro transcripts of RNA3 and D-RNA3 on the inoculated leaves of T.expansa in 5 dpi. (B) Northern analysis of wild type RNA3 and D-RNA3 mutants from inoculated leaves of T.expansa. (C) Symptom induced by RNA1 2, alone or combined with in vitro transcripts of RNA3 and D-RNA3α on the inoculated leaves of C. amaranticolor in 5 dpi.
Mentions: To further investigate the function of D-RNA3s, the full-length cDNA clones of the three D-RNA3s were constructed and inoculated on the local host. The classical local host T. expansa of BNYVV could be induced to form faint chlorotic spots by RNA1 and 2 (isolate BN12, Figure 3A). In contrast, the co-infection of full-length RNA3 and BN12 would cause yellow spots in 5 dpi, as described previously (Figure 3A) [17]. The D-RNA 3β or D-RNA 3γ, as did BN12 alone, only caused faint chlorotic spots, demonstrating the deletion form of p25 protein was deficient for inducing yellow spots. Surprisingly, the D-RNA3α induced obvious necrotic spots on T. expansa in 3 dpi, and then some yellow halos were shown around the necrotic spot in the few days later (Figure 3A). In the C. amaranticolor, the D-RNA3α could also induce necrotic spot in the inoculated leaves (Figure 3C). The northern results showed the D-RNA3s form could be replicated as efficiently as wild type RNA3, indicating the deletion region had no effect on the replication of RNA3 (Figure 3B).

Bottom Line: Our studies demonstrated the internal deletion mutants of BNYVV-RNA3 were spontaneously generated in the systemic infection on N. benthamiana.The internal deletions didn't affect the efficient replication of D-RNA3s, instead by improving the stability and pathogenicity of RNA3 in the systemic host N. benthamiana.Besides, our results also suggested the downstream N protein of RNA3, but not the upstream p25 protein, may play an important role in the systemic infection on N. benthamiana.

View Article: PubMed Central - HTML - PubMed

Affiliation: State Key Laboratory for Agro-biotechnology and Ministry of Agriculture Key Laboratory for Plant Pathology, China Agricultural University, Beijing 100193, PR China.

ABSTRACT

Background: Beet Necrotic Yellow Vein virus (BNYVV) is a member of the genus Benyvirus causing a worldwide sugar beet disease rhizomania. BNYVV contains four or five plus-sense single stranded RNAs. In altered selective conditions, multipartite RNA viruses of plant are prone to undergoing internal deletions, thus turning into Defective RNAs (D RNAs). Although several D RNAs have been reported in BNYVV infection, the spontaneous internal deletion mutants responsible for severe symptom in systemic host Nicotiana benthamiana (N. benthamiana) are not described so far.

Results: Systemic host N. benthamiana was inoculated by Chinese BNYVV isolates. RT-PCR and Northern blot showed that the D RNAs forms of BNYVV RNA3 were present in the systemic infection of the N. benthamiana. Three distinct D-RNA3s, named as D-RNA 3α, D-RNA 3β and D-RNA 3γ, were made into infectious clones. When inoculated on the N. benthamiana, the in vitro transcripts of D forms exhibited more stable than that of wild-type RNA3 in systemic movement. Among the detected mutant, the p25 protein frame-shift mutant (D-RNA3α) induced obvious necrotic lesions on Tetragonia.expansa (T. expansa) and pronounced systemic symptom on the N. benthamiana. The D-RNA3α was further mutated artificially to pre-terminate the downstream N protein, leading to the abolishment of the pathogenicity, indicating the N protein was responsible for the necrotic symptom.

Conclusion: Our studies demonstrated the internal deletion mutants of BNYVV-RNA3 were spontaneously generated in the systemic infection on N. benthamiana. The internal deletions didn't affect the efficient replication of D-RNA3s, instead by improving the stability and pathogenicity of RNA3 in the systemic host N. benthamiana. Besides, our results also suggested the downstream N protein of RNA3, but not the upstream p25 protein, may play an important role in the systemic infection on N. benthamiana.

Show MeSH
Related in: MedlinePlus