Journal of Experimental Botany, Vol. 53, No. 375, pp. 1831-1832,
August 1, 2002
© 2002 Oxford University Press
Dodder infection induces the expression of a pathogenesis-related gene of the family PR-10 in alfalfa
Received 17 April 2002; Accepted 13 May 2002
Agricultural Biotechnology Center, PO Box 411, H-2101 Gödöllõ, Hungary
1 To whom correspondence should be addressed. Fax: +36 28 430416. E-mail: borsics{at}abc.hu
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Abstract |
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A full-length cDNA, PPRG2, representing a gene highly expressed in dodder (Cuscuta trifolii Bab et. Gibs)-infected alfalfa (Medicago sativa L.) stems was isolated by differential screening. The predicted protein contains 157 amino acids and belongs to the PR-10 family of the pathogenesis-related genes with putative ribonuclease activities. Northern hybridizations showed that PPRG2 is transcribed in root and crops of uninfected alfalfa and is induced not only by dodder attack but also by bacterial infections and a large variety of environmental stresses.
Key words: Key words: Cuscuta, Medicago, parasitism, pathogenesis-related proteins, stress.
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Dodder (Cuscuta spp) is a twining yellow holoparasitic plant that parasitizes various kinds of wild and cultivated plants. Like all parasites, dodder grows haustoria, a modified adventitious root, that penetrates the host’s tissue and absorbs water, minerals and carbohydrates from the host. Touch and penetration of haustoria, removal of the resources from the host and the subsequent hormonal changes are all able to trigger host defence responses including alterations in gene expression.
Previous studies showed that the promoters of a defence-related isogene of 3-hydroxy-3-methylglutaryl CoA reductase (HMGR) and a pathogenesis-related (PR) gene are initiated in tobacco roots upon the attack of the parasitic plant Orobanche (Westwood et al., 1998; Joel and Portnoy, 1998). Gowda et al. (1999) described a gene (NRSA1) related to disease resistance genes that is induced during the non-compatible interaction of the marigold and the root parasite witchweed. The first gene (PPRG1) isolated from a compatible host–parasite interaction was reported in a previous study (Borsics and Lados, 2001). PPRG1 is derived from alfalfa and encodes a putative calmodulin-related protein.
The cDNA clone PPRG1 was obtained by differential display. To isolate novel genes highly expressed in plant–parasite interaction, a cDNA library was established from the alfalfa–dodder parasitic region and the technique of differential screening was applied (Sambrook et al., 1989). This resulted in the isolation of 13 clones. Six of them were positive for the parasitic region when tested by reverse Northern hybridization. Two clones showed cross-hybridization. Sequence comparison of the two clones resulted in the detection of four mismatches, two of them locating in the coding region without alterations in the amino acid sequence. However, both cDNAs were truncated at their 5' region. Nested primers 5'-GTA ATT AGG GTT TGC CAC GAC G-3' (primer 1) and 5'-CCC TGT TCC TCC TAC CAA GC-3' (primer 2) were used to obtain the remaining part of the corresponding gene. The full-length cDNA encoding a protein of 157 aa was designated PPRG2 (accession number AJ320476).
Induction of PPRG2 expression in the parasitic region was verified by Northern hybridization (Fig. 1A). The PPRG2 transcript became apparent by day 3 after infection and remained at high level at least for 2 weeks in the infected region (Fig. 1B). Southern blot analysis showed that PPRG2 cDNA was derived from alfalfa (Fig. 1C). Expression of PPRG2 is organ-specific. High amounts of steady-state PPRG2 mRNA were found in the root and crop while a low mRNA level was detected in stem, leaf and flower (Fig. 1D).
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PPRG1 is inducible by a large variety of environmental stresses (Borsics and Lados, 2001). Figure 1E shows that PPRG2 is also inducible by various abiotic stresses. The mechanical impacts touch, rain and wound, but not wind, result in transient elevation of the PPRG2 mRNA level. Cold (4 °C), heat (42 °C), osmotic (10, 20 or 30% PEG), and salt (2M NaCl) stresses also increased the PPRG2 mRNA level after 2–3 h. However, out of the phytohormones abscisic acid (ABA), 2,4-D, kinetin, methyl-jasmonate (MeJA), and SA, only ABA and MeJA influenced PPRG2 transcription.
Computer-assisted analysis of the PPRG2 protein showed substantial homology to many PR-10 proteins from several legume species. Moreover, PPRG2 possesses a Betv1 motif in the third quarter of the protein (88–120 aa) characteristic for the PR-10 family (Breiteneder et al., 1989; van Loon et al., 1994). This finding suggests that like other PR-10 proteins with Betv1 motifs (Bufe et al., 1996), PPRG2 may have ribonuclease activity.
Pathogenesis-related genes were discovered and featured upon their induction by pathogen attack. Expression of PPRG2 in alfalfa leaves was investigated after an incompatible (Pseudomonas) and a compatible (Xanthomonas) pathogen challenge. Both infections resulted in rapid and constant increase in mRNA level (Fig. 1F). Thus PPRG2 is not specific for a certain type of pathogen attack, but like SRG1 (Truesdell and Dickman, 1997), it may be involved in a general plant response to bacterial infections.
Two alfalfa genes, PPRG1 (Borsics and Lados, 2001) and PPRG2 (this work), highly expressed in dodder-infected alfalfa stem have been isolated and characterized so far. Besides dodder infection, both genes are inducible by mechanical impacts. Moreover, PPRG2 belongs to a family of pathogenesis-related genes and is inducible by bacterial infections. These data suggest that common factors in abiotic and biotic stresses including plant–parasite interactions may exist.
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Acknowledgements |
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This work was supported by grants from the FVM (NEM 2-36/1999), the OM (FKFP0029/97) and OMFB (92-97-16-0122). The authors wish to thank Z Bánfalvi for lively and enlightening discussions during the preparation of the manuscript and Professor R Esnault for providing the bacterial strains.
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