Analysis of cis-regulatory elements in differential expression of the PR10 multigene family in pea (Pisum sativum)

Abstract

Genomic copies of 'PR10.1' and 'PR10.3', two members of the 'PR10' multigene family in 'Pisum sativum', were screened for 'cis'-regulatory elements associated with differential expression upon fungal and chemical challenges. Gel shift assays revealed that nuclear proteins from fungus-treated tissues specifically bound two major binding regions in both 'PR10.1' and 'PR10.3'. Deletion analysis of the 'PR10.1' promoter region from -284 to 79 identified two binding sequences, PDA1 and PDA2. PDA1 reacted with all tested nuclear extracts while PDA2 was only bound by extracts from pods treated with the non-pathogenic fungus ' Fusarium solani' f. sp. 'phaseoli' (Fsph) or salicylic acid (SA). Competition assays with oligonucleotides identified two distinct binding sites, PDA2a and PDA2b within PDA2. Similarly, analysis of the ' PR10.3' promoter from -621 to -196 identified a specific binding sequence, PDC1, from -544 to -461. PDC1 reacted strongly with Fsph and SA treatments and weakly with the pathogenic fungus ' F. solani' f. sp. 'pisi' (Fsp) treatment. Database comparisons of oligonucleotide frequencies between 'PR10' genes and other defense genes, and between defense genes and genes not associated with defense, identified 4 conserved motifs, which were present in PDA1, PDA2 and PDC1. Expression of 'PR10.1' and 'PR10.3' was investigated in a time course up to 48 h after challenges. The highest binding activities occurred 2-4 h after challenge, while 'PR10.1' mRNA accumulation did not peak until 8-12 h.p.i. ' PR10.3' was not expressed in pea pods with any treatment, but was expressed in healthy roots. 'PR10.1' expression remained strong up to 48 h.p.i. with the Fsph, treatment, while expression declined after 12 h with the Fsp treatment. These data suggest that PDA2 could play a role in fungus-induced gene expression. The different expression patterns between ' PR10.1' and 'PR10.3' suggested that there is distinct different gene expression regulation among members of the 'PR10' multigene family in pea.