Spatial and temporal accumulation of defense gene transcripts in bean (Phaseolus vulgaris) leaves in relation to bacteria-induced hypersensitive cell death.

Abstract

Primary leaves of 7- to 9-day-old Red Mexican bean plants were inoculated with virulent or avirulent isolates of Pseudomonas syringae pv. phaseolicola, or saprophytic P. fluorescens either by vacuum infiltration of the whole leaf lamina, or by syringe-inoculation of selected leaf panels. In the incompatible combination, resistance was associated with a hypersensitive response (HR). Syringe-inoculated leaves were sampled in three zones: zone 1, the inoculated leaf area; zone 2, the surrounding 0.5-0.7 cm of leaf tissue; and zone 3, the remainder of the leaf. Northern blots of RNA from zones 1, 2, and 3 were probed with bean cDNAs for phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), chitinase (CHT), and lipoxygenase (LOX). Accumulation of PAL, CHS, and CHT transcripts was more rapid and generally of greater magnitude in the incompatible than in the compatible interaction and, in both cases, was observed essentially only in zone 1 tissues. Similarly, antibacterial phytoalexins were only detected in zone 1 from the incompatible interaction. Young primary leaves have a background level of LOX transcripts, which declines as leaves age. This decline was accelerated over the first 12 hr postinoculation (hpi) with avirulent bacteria, whereas a weak transient induction, peaking at 5-6 hpi, was observed in the compatible interaction. A subsequent, strong accumulation of LOX transcripts was seen in both the compatible and incompatible interactions outside the inoculation site starting about 14 hpi. LOX transcripts did not accumulate at the inoculation site itself in the incompatible interaction compared to a relatively strong induction in the compatible interaction. Interestingly, inoculation of leaves with cells of the saprophyte P. fluorescens also induced the accumulation of transcripts for CHS, CHT, and LOX, but generally to a lesser degree than in the incompatible interaction. No HR occurred and no macroscopic cell damage was apparent in leaves inoculated with P. fluorescens. However, at the microscopic level individual, trypan blue-stained, necrotic plant cells were visible. In spite of this and the accumulation of CHS transcripts, no phytoalexin accumulation was found up to 48 hr after inoculation. The spatial and temporal relationship of the hypersensitive reaction to defense gene transcript and phytoalexin accumulation is discussed.