MolGenGenet (1991) 230:113-119 © Springer-Verlag 1991 Pathogenesis-related protein 4 is structurally homologous to the carboxy-terminal domains of hevein, Win-1 and Win-2 Leslie Friedrich, Mary Moyer, Eric Ward, and John Ryals Agricultural BiotechnologyResearch Unit, CIBA-GEIGY Corporation, P.O. Box 12257 Research Triangle Park, NC, 27709-2257, USA Received May 21, 1991 Summary. The extracellular, acidic pathogenesis-related protein, PR-4, was purified to homogeneity from leaves of Nicotiana tabacum infected with tobacco mosaic virus (TMV) and characterized by partial amino acid sequenc- ing. Complementary DNA clones encoding PR-4 were isolated using an oligonucleotide probe based on the sequence of one of the peptides. The deduced PR-4 pro- tein sequence was found to be related to a family of proteins including hevein and Win-l, which have an ami- no-terminal lectin domain and a carboxy-terminal do- main of unknown function. PR-4 is homologous to the carboxy-terminus of these proteins but does not contain the lectin domain. Thus, the organization of the PR-4 family of proteins is similar to that of the plant chitinase family, in that both contain structural subclasses charac- terized by the presence or absence of an amino-terminal lectin domain. This observation is consistent with the proposal that the DNA encoding the lectin domain may be capable of transposing to form new genes encoding proteins of more complex, multi-domain structure. The expression of PR-4 mRNA was found to increase dra- matically in response to TMV infection and the time course of RNA accumulation was similar to that of other PR proteins. Key words: cDNA cloning - Tobacco - Defense genes - Lectins Introduction When phytopathogenic bacteria, fungi or viruses infect plants, the plant may respond by forming a necrotic lesion surrounding the infection site. The biochemical basis of this hypersensitive reaction (HR) is unknown but it functions to limit the systemic spread of the patho- gen (Fritig et al. 1987; Van Loon et al. 1990). Along with the HR, other cellular processes occur that may reduce primary and secondary pathogen infections, in- Offprint requests to: J. Ryals cluding the production of antibiotic metabolites (e.g. phytoalexins) and the synthesis of extracellular and va- cuolar proteins (Lamb et al. 1989). Among the newly synthesized proteins are enzymes such as chitinase and /%l,3-glucanase that may act directly by hydrolyzing components of the fungal cell wall (Boller 1988), and other enzymes such as peroxidase that may act indirectly by strengthening the plant cell wall, making the tissue less susceptible to infection (Bowles 1990). Other newly synthesized proteins, such as pathogenesis-related (PR) proteins also accumulate in the extracellular spaces of the infected tissue to levels approaching 1% of the total cellular protein (Van Loon 1985; Bol et al. 1990; Carr and Klessig 1990). The PR-proteins were first described as acidic, extra- cellular proteins of relatively low molecular weight that accumulate in tobacco leaves reacting hypersensitively to tobacco mosaic virus (TMV) infection (Van Loon and Van Kammen 1970; Gianinazzi et al. 1970). In tobacco, 10 PR-proteins, originally designated PR-la, -lb, -lc, -2, -N, -O, -P, -Q, -R, and -S have been described, which migrate with decreasing mobility on non-denaturing polyacrylamide gels (Van Loon et al. 1983). Recently, a change in nomenclature has been suggested based on the serological relationships between the proteins (Van Loon et al. 1987). The designations PR-la, PR-Ib and PR-lc remain the same; PR-2, -N and -O are now termed PR-2a, PR-2b and PR-2c, respectively; PR-P and PR-Q become PR-3a and PR-3b, respectively; PR-R be- comes PR-4; and PR-S becomes PR-5. In this report we will refer to the proteins using this new nomenclature. While no activity has been described for either the PR-I or PR-4 proteins, it has been demonstrated that the PR-2 family of proteins have in vitro/%l,3-glucanase activity (Kauffmann et al. 1987), PR-3 proteins have chitinase activity (Legrand et al. 1987) and the PR-5 proteins are structurally similar to an alpha-amylase/trypsin inhibi- tor from maize (Cornelissen et al. 1986; Pierpoint et al. 1987; Richardson et al. 1987). In recent years, cDNA and genomic clones encoding PR-1, PR-2, PR-3 and PR-5 have been isolated, which has facilitated studies of the molecular biology of the