Role of calcium in signal transduction during the hypersensitive response caused by basidiospore-derived infection of the cowpea rust fungus

The hypersensitive response (HR) of disease-resistant plant cells to fungal invasion is a rapid cell death that has some features in common with programmed cell death (apoptosis) in animals. We investigated the role of cytosolic free calcium ([Ca2+]i) in the HR of cowpea to the cowpea rust fungus. By using confocal laser scanning microscopy in conjunction with a calcium reporter dye, we found a slow, prolonged elevation of [Ca2+]i in epidermal cells of resistant but not susceptible plants as the fungus grew through the cell wall. [Ca2+]i levels declined to normal levels as the fungus entered and grew within the cell lumen. This elevation was related to the stage of fungal growth and not to the speed of initiation of subsequent cell death. Elevated [Ca2+]i levels also represent the first sign of the HR detectable in this cowpea-cowpea rust fungus system. The increase in [Ca2+]i was prevented by calcium channnel inhibitors. This effect was consistent with pharmacological tests in which these inhibitors delayed the HR. The data suggest that elevation of [Ca2+]i is involved in signal transduction leading to the HR during rust fungal infection.     

Involvement of reactive oxygen species in the response of resistant (hypersensitive) or susceptible cowpeas to the cowpea rust fungus

A previous study had indicated that scavengers of reactive oxygen species (ROS) delayed cell death (the hypersensitive response (HR)) triggered in epidermal cells of intact, resistant, cowpea ( Vigna unguiculata (L.) Walp) leaves by the monokaryotic stage of the cowpea rust fungus ( Uromyces vignae Barclay race 1). This HR had been monitored by cell autofluorescence, which occurs after protoplast collapse. In the present study, when cytoplasmic disorganization was used to monitor cell death more directly, ROS-scavengers, superoxide dismutase, catalase, horseradish peroxidase, and desferal-Mn(IV) had no effect on HR development. Cytological staining for superoxide or hydrogen peroxide generation also did not reveal the presence of ROS before or during the early stages of the HR, but did, as in the previous study, suggest a role in the autofluorescence and browning of invaded cells that occur following protoplast collapse. Staining of plant mitochondria with nitroblue tetrazolium, possibly attributable to increased dehydrogenase activity but not superoxide generation, occurred transiently around invasion hyphae (monokaryotic stage of the fungus) or haustoria (dikaryotic stage) of the fungus as they entered a cell in the susceptible or resistant cultivar. Around invasion hyphae in epidermal cells in resistant plants, this staining diminished as cytoplasmic streaming stopped, and gradually disappeared as cell death progressed. These data are consistent with other evidence that rust fungi initially negate non-specific defensive responses in both resistant and susceptible cells as part of the establishment of biotrophy. They also suggest that the HR in the cowpea–cowpea rust fungus pathosystem is not triggered by an oxidative burst.     

In vitro screening of taxol,an anticancer drug produced by the fungus,Colletotrichum capsici

The fungus Colletotrichum capsici was isolated from the diseased fruits of Chilli plant, Capsicum annuum. The isolated test fungus was identified by its morphological and molecular characteristic features. For the first time, the fungus was screened for the production of taxol on modified liquid medium. The presence of taxol was confirmed by the spectroscopic and chromatographic methods of analyses. The amount of taxol produced by this fungus was quantified by HPLC. The maximum amount of fungal taxol production was recorded as 687 μg/L. The production rate was 13 740‐fold higher than that, previously reported for the fungus Taxomyces andreanae. The extracted fungal taxol showed a strong cytotoxic activity in an in vitro culture of human cancer cells indicating that the increase in taxol concentration induces increased cell death. A PCR‐based screening for taxadiene synthase (ts), a unique gene in the formation of the taxane skeleton, confirmed the molecular blueprint for taxol biosynthesis. The results show that the fungus C. capsici is an excellent candidate for an alternate source of taxol supply and can serve as a potential species for genetic engineering to enhance the production of taxol to a higher level.     

Cleavage of Nuclear DNA into Oligonucleosomal Fragments during Cell Death Induced by Fungal Infection or by Abiotic Treatments

It is often claimed that programmed cell death (pcd) exists in plants and that a form of pcd known as the hypersensitive response is triggered as a defense mechanism by microbial pathogens. However, in contrast to animals, no feature in plants universally identifies or defines pcd. We have looked for a hallmark of pcd in animal cells, namely, DNA cleavage, in plant cells killed by infection with incompatible fungi or by abiotic means. We found that cell death triggered in intact leaves of two resistant cowpea cultivars by the cowpea rust fungus is accompanied by the cleavage of nuclear DNA into oligonucleosomal fragments (DNA laddering). Terminal deoxynucleotidyl transferase-mediated dUTP nick end in situ labeling of leaf sections showed that fungus-induced DNA cleavage occurred only in haustorium-containing cells and was detectable early in the degeneration process. Such cytologically detectable DNA cleavage was also observed in vascular tissue of infected and uninfected plants, but no DNA laddering was detected in the latter. DNA laddering was triggered by [greater than or equal to]100 mM KCN, regardless of cowpea cultivar, but not by physical cell disruption or by concentrations of H2O2, NaN3, CuSO4, or ZnCl2 that killed cowpea cells at a rate similar to that of ladder-inducing KCN concentrations. These and other results suggest that the hypersensitive response to microbial pathogens may involve a pcd with some of the characteristics of animal apoptosis and that DNA cleavage is a potential indicator of pcd in plants.     

Localization of Ptr ToxA Produced by Pyrenophora tritici-repentis Reveals Protein Import into Wheat Mesophyll Cells

The plant pathogenic fungus Pyrenophora tritici-repentis secretes host-selective toxins (HSTs) that function as pathogenicity factors. Unlike most HSTs that are products of enzymatic pathways, at least two toxins produced by P. tritici-repentis are proteins and, thus, products of single genes. Sensitivity to these toxins in the host is conferred by a single gene for each toxin. To study the site of action of Ptr ToxA (ToxA), toxin-sensitive and -insensitive wheat (Triticum aestivum) cultivars were treated with ToxA followed by proteinase K. ToxA was resistant to protease, but only in sensitive leaves, suggesting that ToxA is either protected from the protease by association with a receptor or internalized. Immunolocalization and green fluorescent protein tagged ToxA localization demonstrate that ToxA is internalized in sensitive wheat cultivars only. Once internalized, ToxA localizes to cytoplasmic compartments and to chloroplasts. Intracellular expression of ToxA by biolistic bombardment into both toxin-sensitive and -insensitive cells results in cell death, suggesting that the ToxA internal site of action is present in both cell types. However, because ToxA is internalized only in sensitive cultivars, toxin sensitivity, and therefore the ToxA sensitivity gene, are most likely related to protein import. The results of this study show that the ToxA protein is capable of crossing the plant plasma membrane from the apoplastic space to the interior of the plant cell in the absence of a pathogen.     

Taxol Determination from Pestalotiopsis pauciseta, a Fungal Endophyte of a Medicinal Plant

Taxol is the most effective antitumor agent developed in the past three decades. It has been used for effective treatment of a variety of cancers. A taxol-producing endophytic fungus Pestalotiopsis pauciseta (strain CHP-11) was isolated from the leaves of Cardiospermum helicacabum and screened for taxol production. The fungus was identified based on the morphology of the fungal culture and the characteristics of the spores and screened for taxol production. The amount of taxol produced by this endophytic fungus was quantified by HPLC and it produced 113.3 mg/L, thus the fungus can serve as a potential material for fungus engineering to improve taxol production. This fungal taxol also had strong anticancer activity against some cancer cells viz., BT 220, H116, Int 407, HL 251 and HLK 210 tested by Apoptotic assay and it is indicated that with the increase of taxol concentration from 0.005–0.05 mmol/L, taxol induced increased cell death through apoptosis.     

Activation of Cysteine Proteases in Cowpea Plants during the Hypersensitive Response-A Form of Programmed Cell Death

There is increasing evidence that the hypersensitive response during plant–pathogen interactions is a form of programmed cell death. In an attempt to understand the biochemical nature of this form of programmed cell death in the cowpea–cowpea rust fungus system, proteolytic activity in extracts of fungus-infected and uninfected cowpea plants was investigated, using exogenously added poly(ADP-ribose) polymerase as a marker. Unlike the proteolytic cleavage pattern of endogenous poly(ADP-ribose) polymerase in apoptotic animal cells, exogenously added poly(ADP-ribose) polymerase in extracts of fungus-infected plants was proteolytically cleaved into fragments of molecular masses 77, 52, 47, and 45 kDa.In vitroandin vivoprotease inhibitor experiments revealed the activation of cysteine proteases, and possibly a regulatory role, during the hypersensitive response.     

Disruption of microtubular cytoskeleton induced by cryptogein, an elicitor of hypersensitive response in tobacco cells

The dynamics of microtubular cytoskeleton were studied in tobacco (Nicotiana tabacum cv Xanthi) cells in response to two different plant defense elicitors: cryptogein, a protein secreted by Phytophthora cryptogea and oligogalacturonides (OGs), derived from the plant cell wall. In tobacco plants cryptogein triggers a hypersensitive-like response and induces systemic resistance against a broad spectrum of pathogens, whereas OGs induce defense responses, but fail to trigger cell death. The comparison of the microtubule (MT) dynamics in response to cryptogein and OGs in tobacco cells indicates that MTs appear unaffected in OG-treated cells, whereas cryptogein treatment caused a rapid and severe disruption of microtubular network. When hyperstabilized by the MT depolymerization inhibitor, taxol, the MT network was still disrupted by cryptogein treatment. On the other hand, the MT-depolymerizing agent oryzalin and cryptogein had different and complementary effects. In addition to MT destabilization, cryptogein induced the death of tobacco cells, whereas OG-treated cells did not die. We demonstrated that MT destabilization and cell death induced by cryptogein depend on calcium influx and that MT destabilization occurs independently of active oxygen species production. The molecular basis of cryptogein-induced MT disruption and its potential significance with respect to cell death are discussed.     

Harpin-induced hypersensitive cell death is associated with altered mitochondrial functions in tobacco cells

Mitochondria play important roles in animal apoptosis and are implicated in salicylic acid (SA)-induced plant resistance to viral pathogens. In a previous study, we demonstrated that SA induces rapid inhibition of mitochondrial electron transport and oxidative phosphorylation in tobacco cells. In the present study, we report that plant programmed cell death induced during pathogen elicitor-induced hypersensitive response (HR) is also associated with altered mitochondrial functions. Harpin, an HR elicitor produced by Erwinia amylovora, induced inhibition of ATP synthesis in tobacco cell cultures. Inhibition of ATP synthesis occurred almost immediately after incubation with harpin and preceded hypersensitive cell death induced by the elicitor. Diphenylene iodonium, an inhibitor of the oxidative burst, did not block harpin-induced inhibition of ATP synthesis or cell death, suggesting that oxidative burst was not the direct cause for these two harpin-induced processes. Unlike SA, harpin had no significant effect on total respiratory O2 uptake of treated cells. However, respiration of harpin-treated tobacco cells became very sensitive to the alternative oxidase inhibitors salicyl-hydroxamic acid and n-propyl gallate. Thus, harpin treatment resulted in reduced capacity of mitochondrial cytochrome pathway electron transport, which could lead to the observed inhibition of ATP synthesis. Given the recently demonstrated roles of mitochondria in apoptosis, this rapid inhibition of mitochondrial functions may play a role in harpin-induced hypersensitive cell death.     

Effect of legume plant density and mixed culture on symbiotic N2 fixation in five cowpea (Vigna unguiculata L. Walp.) genotypes in South Africa

A field experiment involving two plant densities (83,333 and 166,666 plants per hectare), two cropping systems (monoculture and mixed culture) and five cowpea (Vigna unguiculata L. Walp.) genotypes (3 farmer-selected varieties: Bensogla, Sanzie and Omondaw, and 2 breeder-improved cultivars: ITH98-46 and TVuI509) was conducted for two years in 2005 and 2006 at Nietvoorbij (33°54S, 18°14E), Stellenbosch, South Africa, to evaluate the effect of these treatments on the growth and symbiotic performance of cowpea. The results showed that, of the five cowpea genotypes, plant growth and N₂ fixation were significantly greater in the three farmer-selected varieties (Sanzie, Bensogla and Omondaw) relative to the two improved cultivars (ITH98-46 and TVuI509). Furthermore, plant growth and symbiotic performance (measured as tissue N concentration, plant N content,¹⁵N natural abundance and N-fixed) were significantly (P<-50.05) decreased by both high plant density and mixed culture (intercropping). However, the %Ndfa values were significantly (P<-50.05) increased by both high plant density and mixed culture compared to low plant density or monoculture (or monocropping). Whether under low or high plant density, the cv. Sanzie was found to accumulate significantly greater total N per plant in both 2005 and 2006, followed by the other two farmer varieties relative to the improved cultivars. Similarly, the actual amount of N-fixed was much greater in cv. Sanzie, followed by the other farmer varieties, under both low and high plant density. The data also showed better growth and greater symbiotic N yield in cowpea plants cultivated in monoculture (or low plant density) relative to those in mixed culture (or high plant density). Our data suggest that optimising legume density in cropping systems could potentially increase N₂ fixation in cowpea, and significantly contribute to the N economy of agricultural soils in Africa.     

The hypersensitive response and the induction of cell death in plants

The hypersensitive response, or HR, is a form of cell death often associated with plant resistance to pathogen infection. Reactive oxygen intermediates and ion fluxes are proximal responses probably required for the HR. Apoptosis as defined in animal systems is, thus far, not a strict paradigm for the HR. The diversity observed in plant cell death morphologies suggests that there may be multiple pathways through which the HR can be triggered. Signals from pathogens appear to interfere with these pathways. HR may play in plants the same role as certain programmed cell deaths in animals with respect to restricting pathogen growth. In addition, the HR could regulate the defense responses of the plant in both local and distant tissues.     

Cell Death Control: The Interplay of Apoptosis and Autophagy in the Pathogenicity of Sclerotinia sclerotiorum

Programmed cell death is characterized by a cascade of tightly controlled events that culminate in the orchestrated death of the cell. In multicellular organisms autophagy and apoptosis are recognized as two principal means by which these genetically determined cell deaths occur. During plant-microbe interactions cell death programs can mediate both resistant and susceptible events. Via oxalic acid (OA), the necrotrophic phytopathogen Sclerotinia sclerotiorum hijacks host pathways and induces cell death in host plant tissue resulting in hallmark apoptotic features in a time and dose dependent manner. OA-deficient mutants are non-pathogenic and trigger a restricted cell death phenotype in the host that unexpectedly exhibits markers associated with the plant hypersensitive response including callose deposition and a pronounced oxidative burst, suggesting the plant can recognize and in this case respond, defensively. The details of this plant directed restrictive cell death associated with OA deficient mutants is the focus of this work. Using a combination of electron and fluorescence microscopy, chemical effectors and reverse genetics, we show that this restricted cell death is autophagic. Inhibition of autophagy rescued the non-pathogenic mutant phenotype. These findings indicate that autophagy is a defense response in this necrotrophic fungus/plant interaction and suggest a novel function associated with OA; namely, the suppression of autophagy. These data suggest that not all cell deaths are equivalent, and though programmed cell death occurs in both situations, the outcome is predicated on who is in control of the cell death machinery. Based on our data, we suggest that it is not cell death per se that dictates the outcome of certain plant-microbe interactions, but the manner by which cell death occurs that is crucial.     

Cellular differentiation and host invasion by the rice blast fungus Magnaporthe grisea

This review describes current advances in understanding the biology of plant infection by the rice blast fungus Magnaporthe grisea. Development of the specialized infection structure, the appressorium, in M. grisea has recently been shown to be controlled by cell cycle progression and initiation of autophagic, programmed cell death in the fungal spore. Re-cycling of the contents of the fungal spore and peroxisomal fatty acid beta-oxidation are therefore important processes for appressorium function. Following entry to the host plant, new evidence suggests that M. grisea grows biotrophically within rice cells, bounded by the plant plasmalemma, and the fungus moves from cell-to-cell by means of plasmodesmata. Biotrophic proliferation of the fungus is likely to require secretion of effector proteins and suppression of host defences. Consistent with this, a component of the polarized exocytosis machinery of M. grisea is necessary for pathogenicity and also for induction of host defences in an incompatible interaction. Large-scale insertional mutagenesis is now allowing the rapid analysis of gene function in M. grisea, heralding a new approach to the study of this important fungal pathogen.     

Cell death mediated by MAPK is associated with hydrogen peroxide production in Arabidopsis.

Rapid and localized programmed cell death, known as the hypersensitive response (HR) is frequently associated with plant disease resistance. In contrast to our knowledge about the regulation and execution of apoptosis in animal system, information about plant HR is limited. Recent studies implicated the mitogen-activated protein kinase (MAPK) cascade in regulating plant HR cell death as well as several other defense responses during incompatible interactions between plants and pathogens. Here, we report the generation of transgenic Arabidopsis plants that express the active mutants of AtMEK4 and AtMEK5, two closely related MAPK kinases under the control of a steroid-inducible promoter. Induction of the transgene expression by the application of dexamethasone, a steroid, leads to HR-like cell death, which is preceded by the activation of endogenous MAPKs and the generation of hydrogen peroxide. Both prolonged MAPK activation and reactive oxygen species generation have been implicated in the regulation of HR cell death induced by incompatible pathogens. As a result, we speculate that the prolonged activation of the MAPK pathway in cells could disrupt the redox balance, which leads to the generation of reactive oxygen species and eventually HR cell death.     

Role of the arginyl-glycyl-aspartic motif in the action of Ptr ToxA produced by Pyrenophora tritici-repentis

A fundamental problem of plant science is to understand the biochemical basis of plant/pathogen interactions. The foliar disease tan spot of wheat (Triticum aestivum), caused by Pyrenophora tritici-repentis, involves Ptr ToxA, a proteinaceous host-selective toxin that causes host cell death. The fungal gene ToxA encodes a 17.2-kD pre-pro-protein that is processed to produce the mature 13.2-kD toxin. Amino acids 140 to 142 of the pre-pro-protein form an arginyl-glycyl-aspartic (RGD) sequence, a motif involved in the binding of some animal proteins and pathogens to transmembrane receptor proteins called integrins. Integrin-like proteins have been identified in plants recently, but their role in plant biology is unclear. Our model for Ptr ToxA action predicts that toxin interacts with a putative host receptor through the RGD motif. Mutant clones of a ToxA cDNA, created by polymerase chain reaction such that the RGD in the pro-toxin was changed to arginyl-alanyl-aspartic or to arginyl-glycyl-glutamic, were expressed in Escherichia coli. Extracts containing mutated forms of toxin failed to cause host cell death, but extracts from E. coli expressing both a wild-type pro-protein cDNA and a control mutation away from RGD were active in cell death development. In competition experiments, 2 mM RGD tripeptide reduced the level of electrolyte leakage from wheat leaves by 63% when co-infiltrated with purified Ptr ToxA (15 microg mL(-1)) obtained from the fungus, but the control peptide arginyl-glycyl-glutamyl-serine provided no protection. These experiments indicate that the RGD motif of Ptr ToxA is involved with toxin action, possibly by interacting with a putative integrin-like receptor in the host.     

The plant gene CCD1 selectively blocks cell death during the hypersensitive response to Cauliflower mosaic virus infection

The P6 protein of Cauliflower mosaic virus (CaMV) W260 elicits a hypersensitive response (HR) on inoculated leaves of Nicotiana edwardsonii. This defense response, common to many plant pathogens, has two key characteristics, cell death within the initially infected tissues and restriction of the pathogen to this area. We present evidence that a plant gene designated CCD1, originally identified in N. bigelovii, can selectively block the cell death pathway during HR, whereas the resistance pathway against W260 remains intact. Suppression of cell death was evident not only macroscopically but also microscopically. The suppression of HR-mediated cell death was specific to CaMV, as Tobacco mosaic virus was able to elicit HR in the plants that contained CCD1. CCD1 also blocks the development of a systemic cell death symptom induced specifically by the P6 protein of W260 in N. clevelandii. Introgression of CCD1 from N. bigelovii into N. clevelandii blocked the development of systemic cell death in response to W260 infection but could not prevent systemic cell death induced by Tomato bushy stunt virus. Thus, CCD1 blocks both local and systemic cell death induced by P6 of W260 but does not act as a general suppressor of cell death induced by other plant viruses. Furthermore, experiments with CCD1 provide further evidence that cell death could be uncoupled from resistance in the HR of Nicotiana edwardsonii to CaMV W260.     

Genetic organization of the hrp gene cluster in Acidovorax avenae strain N1141 and a novel effector protein that elicits immune responses in rice (Oryza sativa L.)

The immune system of plants consists of two main arms, pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) and effector-triggered immunity (ETI). The multiple effectors that trigger ETI are translocated into plant cells by the type III secretion system (T3SS) of pathogenic bacteria. The rice-avirulent N1141 strain of Acidovorax avenae causes ETI in rice, including hypersensitive response (HR) cell death. Sequence analysis indicated that the N1141 genome contains the hrp gene cluster (35.3 kb), including genes encoding the T3SS apparatus. The T3SS-defective N1141 mutant (NΔT3SS) did not cause HR cell death, suggesting that ETI is caused by translocation of effector proteins into rice cells via T3SS. Computational sequence analysis predicted that Lrp, HrpW, and HrpY are secreted by T3SS. The hrpY deletion mutant (NΔhrpY) did not cause ETI, suggesting that HrpY is an important effector of ETI in the interaction between A. avenae N1141 and rice.     

Behavioural response of alate Aphis craccivora Koch (Homoptera: Aphididae) to volatiles from different cowpea cultivars

The cowpea aphid, Aphis craccivora, is a major insect pest of cowpea in Africa. Volatile organic compounds (VOCs) mediate plant–arthropod interactions that could be used in the management of insect pests. In this study, we established the VOC profile involved in the interaction between A. craccivora and four cowpea cultivars, namely Ex‐Luanda, Katumani 80, Machakos 66 and Ken Kunde 1. Behavioural assays were conducted to study host preference and gas chromatography‐mass spectrometry (GC/MS) for chemical analysis of volatiles. In preference assays, alate A. craccivora had no significant preference for any of the four cowpea cultivars tested. However, in the olfactometer assays, the aphids showed a significant preference for odours from cultivar Ex‐Luanda compared to Katumani 80. Machakos 66 and Ken Kunde 1 elicited neutral responses. In pairwise comparisons, alate A. craccivora did not distinguish between odours of respective cowpea cultivars. GC/MS analysis identified 23 compounds in the volatiles of the four cowpea cultivars. Not all compounds were detected in all cowpea cultivars, and the detected compounds amounts varied in each cultivar. Of these, only four compounds (hexanal, (E)‐2‐hexenal, 1‐octen‐3‐ol and p‐xylene) were emitted in significantly different quantities in the four cultivars. A blend of hexanal and (E)‐2‐hexenal added to cowpea cultivar Ex‐Luanda decreased its attractiveness to A. craccivora compared to the control. Our findings showed differential attractiveness of VOCs of cowpea cultivars to A. craccivora, suggesting that VOCs could be used in the management of A. craccivora.