Verna J. Higgins

The Role of the Jasmonate Response in Plant Susceptibility to Diverse Pathogens with a Range of Lifestyles

Plants defend themselves against attack from insects and pathogens with various resistance strategies. The jasmonate and salicylate signaling pathways are two induced responses that protect plants against these attackers. Knowledge of the range of organisms that are affected by each response is important for understanding how plants coordinate their defenses against multiple attackers and the generality of effect of different resistance mechanisms. The jasmonate response is known to protect plants against a wide range of insect herbivores; in this study, we examined the role of the jasmonate response in susceptibility to eight pathogens with diverse lifestyles in the laboratory and field. Recent biochemical models suggest that the lifestyle of the pathogen (necrotroph versus biotroph) should predict whether the jasmonate response will be involved in resistance. We tested this by examining the susceptibility of wild-type (cv Castlemart with no known genes for resistance to the pathogens used) and jasmonate-deficient mutant tomato (Lycopersicon esculentum) plants (def1) and by employing rescue treatments of the mutant. Plant susceptibility to five of the eight pathogens we examined was reduced by the jasmonate response, including two bacteria (Pseudomonas syringae and Xanthomonas campestris), two fungi (Verticillium dahliae and Fusarium oxysporum f. sp. lycopersici), and an oomycete (Phytophthora infestans). Susceptibility to three fungi was unaffected (Cladosporium fulvum, Oidium neolycopersici, and Septoria lycopersici). Our results indicate that the jasmonate response reduces damage by a wide range of pathogens from different lifestyles, a result that contrasts with the emerging picture of diseases on Arabidopsis. Thus, the generality of jasmonate-based resistance of tomato challenges the view that ecologically distinct plant parasites are resisted via different mechanisms.

Race-specific elicitors of Cladosporium fulvum promote translocation of cytosolic components of NADPH oxidase to the plasma membrane of tomato cells.

The effect of race-specific elicitors on NADPH oxidase was examined in vivo by treating tomato cells with elicitor-containing intercellular fluids prepared from infected tomato leaves inoculated with specific Cladosporium fulvum races. Treatment of Cf-4 or Cf-5 cells with intercellular fluids from incompatible but not from compatible races of C. fulvum increased oxidase activity and the amount of p67-phox, p47-phox, and rac2 in the plasma membrane. Comparison of these three components in the cytosol and plasma membrane indicated that elicitors promoted the translocation of cytosolic components of NADPH oxidase to the plasma membrane of tomato cells carrying the appropriate resistance gene. Protein kinase C activators and inhibitors did not affect enzyme activity or the binding of these three components to the plasma membrane. In contrast, staurosporine, calmodulin antagonists, and EGTA inhibited elicitor-induced oxidase activity and the translocation of the cytosolic components. The assembly process involves a Ca(2+)-dependent protein kinase that catalyzes the phosphorylation of p67-phox and p47-phox, facilitating their translocation to the plasma membrane. Our data suggest that although both plants and animals share common elements in eukaryotic signal transduction, the involvement of different protein kinases mediating the activation of phosphorylation of p67-phox and p47-phox may reflect the unique spatial and temporal distribution of signal transduction pathways in plants.

Plant Defense Response to Fungal Pathogens (Activation of Host-Plasma Membrane H+-ATPase by Elicitor-Induced Enzyme Dephosphorylation).

Elicitor preparations containing the avr5 gene products from race 4 of Cladosporium fulvum and tomato (Lycopersicon esculentum L.) cells near isogenic for the resistance gene Cf5 were used to investigate events following the treatment of host plasma membranes with elicitor. A 4-fold increase in H+-ATPase activity, coincident with the acidification of the extracellular medium, was detected immediately after elicitor treatment. The elicitor-induced stimulation of the plasma membrane H+-ATPase was inhibited by okadaic acid but not by staurosporine, suggesting that protein dephosphorylation was required for increased H+-ATPase activity. This observation was confirmed by [gamma]-32P labeling and immunodetection of the plasma membrane H+-ATPase. Effects of guanidine nucleotide analogs and mastoparan on the ATPase activity suggested the role of GTP-binding proteins in mediating the putative elicitor-receptor binding, resulting in activation of a phosphatase(s), which in turn stimulates the plasma membrane H+-ATPase by dephosphorylation.

Activation of Plant Plasma Membrane Ca2+-Permeable Channels by Race-Specific Fungal Elicitors

The response of plant cells to invading pathogens is regulated by fluctuations in cytosolic Ca2+ levels that are mediated by Ca2+-permeable channels located at the plasma membrane of the host cell. The mechanisms by which fungal elicitors can induce Ca2+ uptake by the host cell were examined by the application of conventional patch-clamp techniques. Whole-cell and single-channel experiments on tomato (Lycopersicon esculentum L.) protoplasts revealed a race-specific fungal elicitor-induced activation of a plasma membrane Ca2+-permeable channel. The presence of the fungal elicitor resulted in a greater probability of channel opening. Guanosine 5[prime]-[[beta]-thio]diphosphate, a GDP analog that locks heterotrimeric G-proteins into their inactivated state, abolished the channel activation induced by the fungal elicitor, whereas guanosine 5[prime][[gamma]-thio]triphosphate, a nonhydrolyzable GTP analog that locks heterotrimeric G-proteins into their activated state, produced an effect similar to that observed with the fungal elicitor. Mastoparan, which stimulates GTPase activity, mimicked the effect of GTP[[gamma]]S. The addition of HA1004 (a protein kinase inhibitor) in the presence of the elicitor totally abolished channel activity, whereas okadaic acid (a protein phosphatase inhibitor) moderately enhanced channel activity, suggesting that the activation of the channel by fungal elicitors is modulated by a heterotrimeric G-protein-dependent phosphorylation of the channel protein.

Regulation of Plant Defense Response to Fungal Pathogens: Two Types of Protein Kinases in the Reversible Phosphorylation of the Host Plasma Membrane H+-ATPase

The role of reversible phosphorylation of the host plasma membrane H+-ATPase in signal transduction during the incompatible interaction between tomato cells and the fungal pathogen Cladosporium fulvum was investigated. Tomato cells (with the Cf-5 resistance gene) or isolated plasma membranes from Cf-5 cells treated with elicitor preparations from race 2.3 or 4 of C. fulvum (containing the avr5 gene product) showed a marked dephosphorylation of plasma membrane H+-ATPase. Similar treatment with elicitor preparations from races 5 and 2.4.5.9.11 (lacking the avr5 gene product) showed no change in dephosphorylation. Elicitor (race 4) treatment of cells, but not of isolated plasma membranes, for 2 hr resulted in rephosphorylation of the ATPase via Ca2+-dependent protein kinases. The initial (first hour) rephosphorylation was enhanced by protein kinase C (PKC) activators and was prevented by PKC inhibitors. Activity of a second kinase appeared after 1 hr and was responsible for the continuing phosphorylation of the H+-ATPase. This latter Ca2+-dependent kinase was inhibited by a calmodulin (CaM) antagonist and by an inhibitor of Ca2+/CaM-dependent protein kinase II. The activation of the Ca2+/CaM-dependent protein kinase depended on the prior activation of the PKC-like kinase.

Climate change and plant diseases in Ontario

Current models predict that expected climate change in Ontario will significantly affect the occurrence of plant diseases in agriculture and forestry in the coming years. Direct, multiple effects on the epidemiology of plant diseases are expected, including the survival of primary inoculum, the rate of disease progress during a growing season, and the duration of epidemics. These effects will positively or negatively influence individual pathogens and the diseases they cause. Changes in the spectra of diseases are also anticipated. Abiotic diseases associated with environmental extremes are expected to increase, and interactions between biotic and abiotic diseases might represent the most important effects of climate change on plant diseases. The management of plant diseases will also be affected. In agriculture, plant breeding programs are expected to adapt many crops to increased duration of growing seasons and, concurrently, to develop drought and stress tolerance. There will be opportunities for new crops and cultivars to be introduced, but effective systems must be in place to detect new pathogens and prevent them from entering with these new crops. Because of the long-lived nature of trees, forests are slow to adapt, and the impact of climate change will have to be considered in forest management plans. Adaptations in agriculture and forestry have been occurring in Ontario for over 100 years, but these may need to occur at an accelerated rate because of rapid changes in climate. It is critical that the infrastructure of agricultural and forestry research remains strong to ensure successful transition and adaptation.

Mitochondrial alternative oxidase is not a critical component of plant viral resistance but may play a role in the hypersensitive response.

Transgenic tobacco (Nicotiana tabacum) with altered levels of mitochondrial alternative oxidase (AOX) were used to examine the potential role of this electron transport chain protein in resistance to tobacco mosaic virus. We examined the effect of AOX expression on the salicylic acid-induced resistance in susceptible plants and the resistance responses of plants harboring the N-gene. A lack of AOX did not compromise the ability of salicylic acid treatment to heighten the resistance of susceptible plants. In plants with the N-gene, a lack of AOX did not compromise the ability of the hypersensitive response to restrict the virus or the ability of the plant to develop systemic acquired resistance. Overexpression of AOX did not heighten the resistance of susceptible plants, but did result in smaller hypersensitive response lesions, suggesting a link between mitochondrial function and this programmed cell death event. We conclude that AOX is not a critical component of the previously characterized salicylhydroxamic acid-sensitive pathway important in viral resistance.

Plant Defense Response to Fungal Pathogens (II. G-Protein-Mediated Changes in Host Plasma Membrane Redox Reactions).

Elicitor preparations containing the avr5 gene products from races 4 and 2.3 of Cladosporium fulvum, and tomato (Lycopersicon esculentum L.) cells containing the resistance gene Cf5 were used to investigate the involvement of redox processes in the production of active oxygen species associated with the plant response to the fungal elicitors. Here we demonstrate that certain race-specific elicitors of C. fulvum induced an increase in ferricyanide reduction in enriched plasma membrane fractions of tomato cells. The addition of elicitors to plasma membranes also induced increases in NADH oxidase and NADH-dependent cytochrome c reductase activities, whereas ascorbate peroxidase activity was decreased. These results suggest that changes in the host plasma membrane redox processes, transferring electrons from reducing agents to oxygen, could be involved in the increased production of active oxygen species by the race-specific elicitors. Our results also show that the dephosphorylation of enzymes involved in redox reactions is responsible for the race-specific induced redox activity. The effects of guanidine nucleotide analogs and mastoparan on the activation of plasma membrane redox reactions support the role of GTP-binding proteins in the transduction of signals leading to the activation of the defense response mechanisms of tomato against fungal pathogens.

Rôle of the phytoalexin medicarpin in three leaf spot diseases of alfalfa

Abstract The possible role of medicarpin in limiting the size of lesions produced by foliar pathogens of alfalfa was investigated using Stemphylium botryosum, Phoma herbarum var. medicaginis and Leptosphaerulina briosiana. Medicarpin was extracted from alfalfa leaves infected with each of these pathogens after inoculation by spraying whole plants or by using the drop diffusate technique with excised leaves. Medicarpin was present in diffusate solutions only when P. herbarum var. medicaginis was used. In contrast to results obtained in drop diffusate experiments using the corn pathogen Helminthosporium turcicum, the amount of medicarpin accumulating in tissue infected by the alfalfa pathogens was relatively low. Mycelial growth of these three alfalfa pathogens and also of a fourth pathogen, Colletotrichum trifolii, was only slightly inhibited (0 to 12%) in V-8 agar containing medicarpin at a concentration of 75 μg/ml while H. turcicum was inhibited by 50% at between 25 and 50 μg/ml. P. herbarum var. medicaginis and L. briosiana caused a loss of medicarpin when it was added to cultures growing in Czapek medium. The first detectable degradation product formed by P. herbarum var. medicaginis was similar to the degradation product SB I formed by S. boryosum. This same compound was also present in diffusate solutions obtained using P. herbarum var. medicaginis. Preliminary evidence suggested that degradation of medicarpin by L. briosiana involved a constitutively produced enzyme or enzyme complex. The results do not eliminate the possibility that medicarpin plays a role in limiting lesion development in diseases caused by fungi pathogenic to alfalfa. A final conclusion must await data on the localization and concentration of medicarpin in the lesion area.

In vitro and in vivo conversion of phaseollin and pisatin by an alfalfa pathogen Stemphylium botryosum

Abstract The alfalfa pathogen, Stemphylium botryosum , was shown to alter, and possibly degrade, two phytoalexins, pisatin and phaseollin, found in non-host plants. In vitro rates of breakdown depended on phytoalexin concentration and size of inoculum; maximum rates were, however, much lower than those reported for medicarpin, the phytoalexin from alfalfa. Mycelial growth in bioassays was initially inhibited by more than 10 μg/ml of either non-host phytoalexin, but ED 50 values increased markedly during incubation. The first conversion product formed from pisatin or phaseollin was as inhibitory to fungal growth as the parent phytoalexin but each was itself broken down during further incubation of mycelial cultures. The phytoalexins and their first conversion products were detected in pod diffusates and in infected tissues from detached leaves. However, assuming a localized distribution around each infection site, enough pisatin or phaseollin was present to account, theoretically, for the observed cessation of fungal growth.