Session IV: Resistance mechanisms, cellular signalling
Understanding plant disease resistance: dissecting the pathway to
hypersensitive cell death
In Arabidopsis thaliana the RPM1 disease resistance gene governs recognition of Pseudomonas syringae strains (a causal agent of leaf spotting disease) carrying either of the corresponding avirulence gene, avrRpm1 or avrB. Our research uses the avrRpm1/RPM1 interaction to elucidate the signalling hierarchies associated with hypersensitive cell death by using a combination of aequorin-mediated bioluminescence, 2-hybrid analysis and AFLP-cDNA display.
Early responses to microbial challenge: Using aequorin as a calcium reporter, we have also been able to measure, both temporally and quantitatively one of the earliest biochemical events associated with the HR, an increase in cytosolic calcium. However, prior to this activated calcium increase we have demonstrated the host plant is actively responding to pathogen presence in an R gene independent manner. This response may be analogous to the innate immune response, an ancient defense system utilised by mammals, plants and insects. We have identified over 350 AFLP fragments up regulated within the first 120 min after pathogen challenge. The dynamic response at the level of transcription suggests plants respond immediately to the presence of non-self by activation of stereotyped receptors. These receptors probably recognise similar exogenous microbial molecular patterns or endogenous molecules, potentiating immunity through a complex network of molecular and cellular innate pathways. Such pathways probably involving stress-related responses in addition to exogenous microbial molecular patterns.
RPM1 interacting proteins: RPM1 belongs to the largest class of plant disease resistance genes, containing both a nucleotide-binding domain and leucine rich repeats. Recently it has emerged that R genes encode a region of extensive amino acid sequence similarity to the nematode CED-4, mammalian Apaf-1 and FLASH proteins which function as adaptor proteins involved in the activation of the apoptotic proteases CED-3, caspase-9 and caspase 8 respectively. Such parallels at the molecular level and phenotypic level (the hypersensitive response has hallmarks of apoptosis) are intriguing and suggest the possibility that this class of R genes may constitute integral components of a plant apoptosome. We have used this Ap-ATPase domain in 2-hybrid studies and our recent results will be presented.
Plant recognition of Xanthomonas effector proteins
Bacterial spot disease on pepper and tomato plants is caused by the gram-negative bacterium Xanthomonas campestris pv. vesicatoria (Xcv). Basic pathogenicity in susceptible plants and recognition in resistant plants is controlled by a type III protein secretion system which is encoded by hrp genes. The Hrp system secretes effector proteins across the bacterial envelope and probably translocates some of them into the plant cell. One of the effector proteins studied in our laboratory is the avirulence protein AvrBs3. AvrBs3 is a 122 kDa protein with a central domain of 17.5 tandem, nearly identical repeat motifs of 34 amino acids. Xcv strains expressing AvrBs3 are specifically recognized by pepper plants carrying the resistance gene Bs3. AvrBs3 recognition occurs inside the plant cell and results in the induction of the hypersensitive reaction (HR). Interestingly, AvrBs3 also has an effect on susceptible pepper and tomato plants, in which hypertrophy is induced. Both HR induction and hypertrophy depends on functional nuclear localization signals and an acidic activation domain in AvrBs3. To elucidate signalling of AvrBs3 in the plant cell we have performed a yeast-interaction trap screen to identify pepper proteins interacting with AvrBs3. Using a cDNA-AFLP approach pepper genes induced by AvrBs3 were isolated from the susceptible plant. In addition, data on the isolation of Bs4, a tomato resistance gene that mediates recognition of an avrBs3 homolog, avrBs3-2, will be discussed.
Signalling mechanisms leading to programmed cell death during the
Hypersensitive Response in Arabidopsis thaliana
Programmed cell death (PCD) is a form of cellular suicide characteristic of the Hypersensitive Response (HR), during which plants challenged by potential microbial pathogens display rapid and localised cell death. The HR is part of a complex suite of active defence responses induced via a range of molecular communications between plant and pathogen, and that requires elaborate host cell signalling mechanisms. We have been using suspension cultures of Arabidopsis thaliana and pathovars of the phytopathogenic bacterium Pseudomonas syringae pv maculicola to elucidate the signalling pathways leading to PCD during plant:bacteria interactions. Treatment of Arabidopsis cells with harpin, a proteinaceous bacterial elicitor secreted by P. syringae pv syringae, results in the rapid generation of H2O2 and PCD; similarly H2O2 treatment also results in PCD. Both harpin and H2O2 treatments lead to the activation of distinct mitogen-activated protein kinases (MAPKs). Inoculation of Arabidopsis suspension cultures with avirulent but not virulent races of Pseudomonas syringae pv maculicola results in PCD, in keeping with the HR responses seen in planta. Moreover, only inoculation with avirulent bacteria induces a specific burst of H2O2 synthesis and nitric oxide (NO) generation. Exogenous NO also induces PCD in Arabidopsis cells. H2O2 and NO-induced PCD appears to be additive, requiring both common and distinct signalling pathways. H2O2 and NO-induced PCD require gene expression: we are currently exploring global changes in gene expression using microarray analysis.