Abstract
A functional cDNA for the resistance gene N from Nicotiana tabacum cv. Samsun NN to Tobacco mosaic virus (TMV) was obtained by reverse transcription polymerase chain reaction. Recombinant proteins containing various domains of the N protein were used in far-Western screening for the N-interacting protein from tobacco cDNA expression libraries. Nucleotide sequence analysis revealed that 199 cDNAs of 217 positive clones screened from 1.6 × 106 phage plaques were related to the 14-3-3 gene family. Phylogenetic analysis indicated that the 14-3-3 proteins were divided into 17 different 14-3-3 isoforms, including hitherto unidentified novel isoforms i-1 and i-2. In vitro binding assays showed that the probes TIR and LRR domains of N protein and helicase (HEL) domain of TMV replicase bound significantly to 14-3-3 isoforms of groups ω and ψ. These domains barely bound to those of group κ and did not bind to that of group ε. The same probes did not bind to any domains of N protein or the viral HEL domain. Northern blotting showed an increase in 14-3-3 isoform h transcripts coinciding with PR-1a induction after TMV inoculation. The 14-3-3 isoform h as a probe had significant binding to the LRR domain of N protein and the viral HEL domain, but there was only minor binding to the TIR domain. These results raise the possibility that 14-3-3 acts as a scaffolding or adaptor between the N protein and the helicase domain to support the interaction between the R gene product and the viral elicitor.
| Original language | English |
|---|---|
| Pages (from-to) | 221-231 |
| Number of pages | 11 |
| Journal | Journal of General Plant Pathology |
| Volume | 70 |
| Issue number | 4 |
| Publication status | Published - 2004 Aug |
| Externally published | Yes |
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Keywords
- 14-3-3
- Helicase
- N gene
- Nicotiana tabacum
- Resistance
- Tobacco mosaic virus
ASJC Scopus subject areas
- Plant Science
Cite this
Members of 14-3-3 protein isoforms interacting with the resistance gene product N and the elicitor of Tobacco mosaic virus. / Konagaya, Ken Ichi; Matsushita, Yasuhiko; Kasahara, Masahiro; Nyunoya, Hiroshi.
In: Journal of General Plant Pathology, Vol. 70, No. 4, 08.2004, p. 221-231.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Members of 14-3-3 protein isoforms interacting with the resistance gene product N and the elicitor of Tobacco mosaic virus
AU - Konagaya, Ken Ichi
AU - Matsushita, Yasuhiko
AU - Kasahara, Masahiro
AU - Nyunoya, Hiroshi
PY - 2004/8
Y1 - 2004/8
N2 - A functional cDNA for the resistance gene N from Nicotiana tabacum cv. Samsun NN to Tobacco mosaic virus (TMV) was obtained by reverse transcription polymerase chain reaction. Recombinant proteins containing various domains of the N protein were used in far-Western screening for the N-interacting protein from tobacco cDNA expression libraries. Nucleotide sequence analysis revealed that 199 cDNAs of 217 positive clones screened from 1.6 × 106 phage plaques were related to the 14-3-3 gene family. Phylogenetic analysis indicated that the 14-3-3 proteins were divided into 17 different 14-3-3 isoforms, including hitherto unidentified novel isoforms i-1 and i-2. In vitro binding assays showed that the probes TIR and LRR domains of N protein and helicase (HEL) domain of TMV replicase bound significantly to 14-3-3 isoforms of groups ω and ψ. These domains barely bound to those of group κ and did not bind to that of group ε. The same probes did not bind to any domains of N protein or the viral HEL domain. Northern blotting showed an increase in 14-3-3 isoform h transcripts coinciding with PR-1a induction after TMV inoculation. The 14-3-3 isoform h as a probe had significant binding to the LRR domain of N protein and the viral HEL domain, but there was only minor binding to the TIR domain. These results raise the possibility that 14-3-3 acts as a scaffolding or adaptor between the N protein and the helicase domain to support the interaction between the R gene product and the viral elicitor.
AB - A functional cDNA for the resistance gene N from Nicotiana tabacum cv. Samsun NN to Tobacco mosaic virus (TMV) was obtained by reverse transcription polymerase chain reaction. Recombinant proteins containing various domains of the N protein were used in far-Western screening for the N-interacting protein from tobacco cDNA expression libraries. Nucleotide sequence analysis revealed that 199 cDNAs of 217 positive clones screened from 1.6 × 106 phage plaques were related to the 14-3-3 gene family. Phylogenetic analysis indicated that the 14-3-3 proteins were divided into 17 different 14-3-3 isoforms, including hitherto unidentified novel isoforms i-1 and i-2. In vitro binding assays showed that the probes TIR and LRR domains of N protein and helicase (HEL) domain of TMV replicase bound significantly to 14-3-3 isoforms of groups ω and ψ. These domains barely bound to those of group κ and did not bind to that of group ε. The same probes did not bind to any domains of N protein or the viral HEL domain. Northern blotting showed an increase in 14-3-3 isoform h transcripts coinciding with PR-1a induction after TMV inoculation. The 14-3-3 isoform h as a probe had significant binding to the LRR domain of N protein and the viral HEL domain, but there was only minor binding to the TIR domain. These results raise the possibility that 14-3-3 acts as a scaffolding or adaptor between the N protein and the helicase domain to support the interaction between the R gene product and the viral elicitor.
KW - 14-3-3
KW - Helicase
KW - N gene
KW - Nicotiana tabacum
KW - Resistance
KW - Tobacco mosaic virus
UR - http://www.scopus.com/inward/record.url?scp=4344703058&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=4344703058&partnerID=8YFLogxK
M3 - Article
AN - SCOPUS:4344703058
VL - 70
SP - 221
EP - 231
JO - Journal of General Plant Pathology
JF - Journal of General Plant Pathology
SN - 1345-2630
IS - 4
ER -