Isolation and nucleotide sequence of a novel cDNA clone encoding the major form of pathogenesis-related protein R

Abstract

Pathogenesis-related (PR) proteins are acid-soluble, protease-resistant proteins which accumulate in the intercellular spaces of many plants as a result of the hypersensitive reaction to a pathogen [10]. The accumulat ion of PR proteins has also been observed in leaves of plants treated with certain chemical inducers such as acetylsalicylic acid and polyacrylic acid [4, 11]. The presence of PR proteins correlates with the onset o f a broad-range systemic resistance [3, 10] which suggests that the PR proteins may have a role in the establishment or maintenence of the resistant state. The predominant PR proteins expressed after TMV infection in tobacco are PRla, lb, lc, -2, -N, -O, -P, -Q, -R and -S [7, 10]. In an effort to isolate cDNA clones for PR proteins, Bol and colleagues at the University of Leiden constructed a cDNA library from TMV-infected tobacco leaves (Nicotiana tabacurn cv Samsun NN) and cDNA clones were isolated from this library that were induced upon TMV infection [2, 5, 6]. One group of these cDNA clones was shown to encode a protein with 65% identity to the sweet-tasting protein, thaumat in [1]. Later it was found that the thaumatin-like protein has about 60% identity to a maize t rypsin/a-amylase inhibitor [9]. Recently, the thaumatin-like protein has been identified as the tobacco pathogenesis-related protein, PR-R [8]. PR-R exists as two isoforms in tobacco, a major and a minor form which are expressed at a ratio of approximately 3:2 [8]. Cornelissen et al. described the isolation and sequence of the minor form of PR-R [1]. Here we present the sequence of a clone isolated from a cDNA library generated from RNA isolated from leaves of TMV-infected N. tabacum cv. Xanthi-nc which encodes the major form of PR-R. The two cDNA clones of PR-R share 95% identity at the nucleic acid level and 98% identity at the level of the encoded amino acid sequence. When 11 cDNA clones were randomly isolated and partially sequenced it was found that 8 code for the major form of PR-R and 3 code for the minor form of PR-R. Our deduced amino acid sequence for the major and minor forms of PR-R agrees with the protein sequence data from Pierpoint et al. [8] at every position except the asparagine residue at position 26 of the major form. There is no obvious reason for this one discrepancy. The DNA sequence for the minor form of PR-R does encode an asparagine at position 26 ([1], our unpublished results); however, the DNA sequence analysis resulting from sequencing both strands of two independent cDNA clones confirms that there is indeed a G at base 166 which would code for an aspartate.