Characterization, expression and evolution of two novel subfamilies of Pinus monticola cDNAs encoding pathogenesis-related (PR)-10 proteins

Proteins of the pathogenesis-related (PR)-10 family are induced in many plants by phytopathogens and environmental stresses. A multi-gene family of PR10 proteins has previously been found in the genome of western white pine (Pinus monticola Dougl. ex D. Don). We isolated two novel subfamilies of PR10 cDNAs (PmPR10-2 and PmPR10-3) from P. monticola that are distinct from other PR10 genes (PmPR10-1.1-1.14) reported from the same species. The PmPR10 proteins are grouped in three subfamilies based on similarity in amino acid sequences. The sequence identities of PmPR10 proteins are much higher among members within a subfamily than among members of different subfamilies (86-99% versus 59-68%). Induction of both PmPR10-2 and PmPR10-3 mRNAs was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) in needles in response to wounding treatment. PmPR10-3 was also expressed in needles during cold acclimation in winter. Transcript levels of both PmPR10-2 and PmPR10-3 were less than the detectable levels of constitutive expression in roots, stems and vegetative shoots, whereas PmPR10-1.10 mRNA of subfamily I was expressed at various levels. Phylogenetic analysis showed that PmPR10 and PR10 proteins from other conifers are grouped within one clade that is distinct from that of angiosperm PR10 proteins. In the conifer monophyletic group, PR 10 sequences diversify into three distinct clusters. Among these three clusters, some PR 10 proteins from single conifer species showed greater divergence distances than sequences from other conifer species, suggesting that, within the conifers, the multi-gene family underwent great diversification during evolution. Based on ratios of nonsynonymous to synonymous nucleotide substitutions (Ka/Ks), we speculate that positive selection resulted in the divergence of PmPR10 subfamilies I and III. Possible mechanisms and significance of PR10 gene evolution are discussed.