Journal of Experimental Botany, Vol. 53, No. 378, pp. 2279-2280,
November 1, 2002
© 2002 Oxford University Press
Cloning of a grapevine Botrytis-responsive gene that has homology to the tobacco hypersensitivity-related hsr203J
Received 25 March 2002; Accepted 9 July 2002
Laboratoire de Biologie et Physiologie Végétales, Université de Reims Champagne-Ardenne, Equipe de Biochimie et Biologie Moléculaire des Plantes, URVVC-EA 2069, UFR Sciences, Moulin de la Housse–BP 1039, F-51687 Reims Cedex 2, France
2 To whom the correspondence should be addressed. Fax: +33 3 26 91 32 68. E-mail: fabienne.baillieul{at}univ-reims.fr
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Abstract |
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A cDNA encoding a putative HSR203J-like protein (BIG8.1) was obtained from total RNA isolated from Botrytis cinerea-infected grapevine leaves using differential display and RACE techniques. Real time RT-PCR analysis confirmed that the level of mRNA corresponding to BIG8.1 increased in grapevine leaves during the infection progress by B. cinerea. No significant change in mRNA level was observed in leaves after UV exposure. This expression pattern suggests that BIG8.1 could be a HR-specific marker in grapevine like hsr203J in tobacco.
Key words: Key words: Botrytis cinerea, hypersensitive response, UV stress, Vitis vinifera.
Botrytis cinerea, the causal agent of grey mould disease, is a necrotrophic fungal pathogen that attacks over 200 different plant species (Jarvis, 1980). No gene-for-gene resistance has been identified in plant–B. cinerea interactions and environmental factors can affect the balance between the plant’s defences and the pathogen’s ability to overcome them (Elad and Evenson, 1995). Govrin and Levine (2000) demonstrated that the necrotic lesions caused by B. cinerea in Arabidopsis and tobacco plants corresponded to the hypersensitive response (HR) death, characterized by nuclear condensation and induction of the HR-specific gene hsr203J. The HR is considered to be a major element of plant disease resistance (Greenberg, 1996) but, with a necrotrophic pathogen like B. cinerea, the HR, instead of confining the pathogen, facilitates its colonization in the plant (Govrin and Levine, 2000).
Hsr (hypersensitivity-related) genes were characterized to be preferentially activated during the HR in tobacco (Marco et al., 1990). In this group, hsr203J was considered to be a HR-specific marker because its expression has been shown to be strongly correlated with programmed cell death in response to different HR-inducing pathogens and elicitors, and also to various cell-death-triggering extracellular agents such as heavy metals (Pontier et al., 1994, 1998). By contrast, only a low level, if any, of hsr203J expression was observed in response to virulent pathogens or elicitors that induce some defence responses, but not cell death. The HSR203J protein was characterized as a carboxylesterase member of the serine hydrolase family (Baudouin et al., 1997).
The defence responses of grapevine (Vitis vinifera L. cv. Chardonnay) towards Botrytis cinerea were studied using a differential display method (Delta Differential Display Kit, Clontech, France) comparing changes in mRNA levels between B. cinerea-infected (6, 15, 24, and 48 h post-inoculation) and non-infected grapevine leaves (see Bézier et al., 2002, for the infection procedure). Among several putative differential cDNAs fragments, a 263 bp cDNA was characterized further. This cDNA, designed as BIG8.1 (Botrytis-induced grapevine) gene, corresponded to a partial open reading frame (ORF) and a 3' untranslated sequence with a polyA track. Based on the sequence of this product a gene-specific primer was designed to amplify the upstream cDNA by 5'-RACE (rapid amplification of cDNAs ends). Finally, the complete ORF was amplified with specific primers (5'-ATGTCCTTTATTGGAGAA GCATC-3' and 5'-CTAAAAGTTATGAATGAACTCACT-3') derived from sequences of the two partial cDNAs and the total RNA from infected leaves as a template. The complete sequence (Accession No. AF487826), derived from the three overlapping cDNAs is 1137 bp long and contains a 933 bp ORF, a 56 bp 5'-flanking sequence and a 148 bp 3'-flanking sequence with a polyA track. The predicted protein consisted of 310 amino acids and had a calculated molecular mass of 34.8 kDa. A comparison of predicted BIG8.1 with protein databases revealed limited, but significant, similarity with different Ser-active hydrolases (Fig. 1) such as hypothetical hydrolase PrMC3 from Pinus radiata (Accession No. AF110333, Walden et al., 1999), carboxylesterases E86 from pea (Accession No. AB026296, Ichinose et al., 2001) and HSR203J from tobacco (Accession No. CAA54393, Baudouin et al., 1997). The significant homology was localized between amino acids 54 and 163 in BIG8.1. Two consensus sequence motifs, GXSXG characteristic of serine hydrolases and HGGF related to the lipase/esterase family (Baudouin et al., 1997) are found. In this interval BIG8.1 shared 72% homology (55% identity) with PrMC3, and 66% and 64% homology with HSR203J and E86, respectively.
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Real-time RT-PCR analysis was carried out to confirm that infection with B. cinerea does increase the level of BIG8.1 expression (Fig. 2). RNA was extracted from leaves infected under laboratory conditions and analysed as previously described (Bézier et al., 2002), except that the quantity of BIG8.1 mRNA was expressed using the standard curve method (User bulletin No. 2, ABI PRISM 7700 sequence detection system). No basal constitutive accumulation of BIG8.1 mRNA was measured in non-treated plants. A weak amount of mRNA (66 molecules) was detected by 6 hpi. The level of accumulation rose slightly by 15 hpi, reached 36x103 molecules by 24 hpi and a maximum of 166x103 by 48 hpi. A slight accumulation was measured in the mock-inoculated plants, but this was negligible compared to the infected plants.
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Grapevine leaves treated with UV light exhibit an increase in the content of the phytoalexin resveratrol (Langcake and McCarthy, 1979) but not necrosis (data not shown). Increases were measured in mRNA levels of various defence responses, such as glucanase (A Bézier, unpublished results), but according to the expression pattern of hsr203J from tobacco which is highly correlated with cell death, UV stress did not significantly increase the mRNA level of BIG8.1 in exposed leaves (data not shown).
These results suggest that B. cinerea induces an HR in grapevine as it does on Arabidopsis and tobacco plants (Govrin and Levine, 2000) and that BIG8.1 could be considered as an HR-specific marker in grapevine.
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Acknowledgements |
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We thank Cathy Hachet for handling the in vitro plantlets. This research was supported by a grant from Europôl’Agro.
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