Electrophysiological Characterization of the Arabidopsis avrRpt2-Specific Hypersensitive Response in the Absence of Other Bacterial Signals

Abstract

The hypersensitive response (HR) is defined as rapid cell collapse at the infection site and often accompanies plant resistance. The physiological processes leading to HR are not well understood. Here, we report an electrophysiological characterization of bacterial HR caused by a single avirulence gene in the absence of other bacterial signals. We used dexamethasone (dex)-inducible transgenic Arabidopsis (Arabidopsis thaliana) plants containing the avrRpt2 gene from Pseudomonas syringae pv tomato. Membrane depolarization in these plants began 1 to 1.5 h after dex application, hours before electrolyte leakage. Progressive depolarization was a sensitive early indicator of HR that occurred only in Arabidopsis leaf cells expressing both avrRpt2 and a functional RPS2 gene. Hyperpolarization of fully depolarized membranes by fusicoccin, a fungal toxin that activates the H 1 -ATPase, indicates that depolarization did not result from a nonfunctional pump or leaky membranes. Depolarization and electrolyte leakage were inhibited in RPS2 plants by the calcium channel blocker LaCl 3 , highly correlating these events and suggesting that Ca 21 entry into cells is required for both. Also correlated were inhibition of depolarization, electrolyte leakage, and HR following salicylic acid pretreatment. In salicylic acid-pretreated RPS2 seedlings, avrRpt2 transcript was produced after dex treatment. However, AvrRpt2 protein accumulation was greatly reduced, suggesting a possible mechanism for inhibition of HR in plants with induced resistance. This experimental system is a very sensitive assay that lends itself to the dissection of physiological processes leading to HR in plants, and provides a baseline for future research within a genetic framework.Plants are resistant to infection by many pathogens. Plants expressing a resistance (R) gene rapidly initiate defense responses following contact with a pathogen that expresses a corresponding avirulence (avr) gene (Flor, 1971). Isogenic plants lacking the R gene fall prey to the pathogen. The hypersensitive response (HR), rapid cell collapse at the site of infection, is often dramatic evidence of resistance, particularly in bacteriainduced HR (Goodman and Novacky, 1994;Heath, 2000).Many defense responses associated with bacteriainduced HR have been described since Klement et al. (1964) first demonstrated the total collapse of nonhost plant leaves following infiltration of pathogenic bacteria. They include ion fluxes, increased salicylic acid (SA) production, membrane depolarization, and pathogenesis-related (PR) gene induction (Goodman and Novacky, 1994;Dangl et al., 1996; HammondKosack and Jones, 1996;Dangl and Jones, 2001). Yet, the mechanism of HR induction is not well understood.The functional significance of bacteria-induced HR also is not clear because defense responses and resistance can occur without HR. For example, systemically acquired resistance, resistance to a broad spectrum of pathogens, can be induced in normally susceptible plants by pathogen infection or application o...

Chloroplast‐generated reactive oxygen species are involved in hypersensitive response‐like cell death mediated by a mitogen‐activated protein kinase cascade

“…Partial repolarization was observed when the light was turned on. These membrane responses are similar to those seen in plants after infiltration with HR‐inducing bacteria, in transgenic plants expressing a DEX‐inducible avrRpt2 construct, and in plants treated with harpin (Pavlovkin and Novacky, 1986; Pike et al. , 1998, 2005).…”

“…Membrane potential has previously been shown to be a very reliable early indicator of HR or HR‐like cell death (Pavlovkin and Novacky, 1986; Pike et al. , 1998, 2005).…”

The grateful dead: calcium and cell death in plant innate immunity

“…(2000) concluded that such a biphasic Ca 2+ elevation occurred in aequorin‐expressing Arabidopsis plants only in the presence of a pathogen with an avr gene; they concluded that the major, second rise in cytosolic Ca 2+ of the biphasic pattern was specifically induced during avr gene product elicitation of ETI. Pike et al . (2005) provided electrophysiological evidence that a pathogen‐induced Ca 2+ influx occurs due to R gene‐mediated signalling as well.…”

Differential Expression of Biphenyl Synthase Gene Family Members in Fire-Blight-Infected Apple ‘Holsteiner Cox’

“…Core fragments of the MdBIS1 to MdBIS4 cDNAs were amplified with gene-specific primer pairs (Table III). Amplification of profilin transcripts served as control for equal RNA template amounts, as used previously for normalization (Pike et al, 2005). PCR was carried out using Taq DNA polymerase (Peqlab).…”