Characterization of a proteinase inhibitor from Brachypodium distachyon suggests the conservation of defence signalling pathways between dicotyledonous plants and grasses

Abstract

SUMMARY Proteinase inhibitors (PIs) are established markers for wound- and especially jasmonate-mediated signalling in dicot species such as tomato and potato. Differential screening of a cDNA library constructed from RNA isolated from wounded leaves of the grass Brachypodium distachyon led to the identification of a proteinase inhibitor gene (Bdpin1). Bdpin1 exhibited the highest homology to the subtilisin/chymotrypsin-inhibiting subgroup of the pin1 class of plant PIs. Northern analyses indicated that Bdpin1 was induced within 6 h at the site of wounding and systemically, by 24 h, thereby providing evidence for long-distance signalling in grasses. Bdpin1 also proved to be more rapidly induced in susceptible than in resistant ecotypes of B. distachyon following challenge with the Rice blast pathogen, Magnaporthe grisea. Screening with chemical signals indicated that Bdpin1 could be induced with MeJA but not with the putative mimic of salicylic acid, benzothiadiazole. Genomic Southern hybridization was consistent with Bdpin1 existing at a single locus, which was isolated following screening of a genomic cosmid library. DNA upstream of the Bdpin1 coding sequence was characterized via fusion to a GUS reporter and was found to confer wound-responsive transcription in B. distachyon and other cereals following biolistic bombardment. Both wound- and TMV-activated Bdpin1-GUS activity was detected in transgenic tobacco. Given that B. distachyon represents an ancestral grass species, our data suggest that there is considerable conservation in defence-associated signalling between dicots and grasses.