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Pathogens and other adverse environmental conditions can trigger endoplasmic reticulum (ER) stress. ER stress signaling increases the expression of cytoprotective ER-chaperones. The inositol-requiring enzyme (IRE1) is one ER stress sensor that is activated to splice the bZIP60 mRNA that produces a truncated transcription factor that activates gene expression in the nucleus. The IRE1/bZIP60 pathway is associated with restricting potyvirus and potexvirus infection. This study shows that the Plantago asiatica mosaic virus (PlAMV) triple gene block 3 (TGB3) and the Turnip mosaic virus (TuMV) 6K2 proteins activate alternative transcription pathways involving the bZIP17, bZIP28, BAG7, NAC089 and NAC103 factors in Arabidopsis thaliana. Using the corresponding knockout mutant lines, we show that bZIP17, bZIP60, BAG7 and NAC089 are factors in reducing PlAMV infection, whereas bZIP28 and bZIP60 are factors in reducing TuMV infection. We propose a model in which bZIP60 and bZIP17 synergistically induce genes restricting PlAMV infection, while bZIP60 and bZIP28 independently induce genes supporting PlAMV infection. Regarding TuMV-green fluorescent protein (GFP) infection, bZIP60 and bZIP28 serve to repress local and systemic infection. Finally, tauroursodeoxycholic acid treatments were used to demonstrate that the protein folding capacity significantly influences PlAMV accumulation.

Cell death can be driven by a genetically programmed signalling pathway known as programmed cell death (PCD). In plants, PCD occurs during development as well as in response to environmental and biotic stimuli. Our understanding of PCD regulation in plants has advanced significantly over the past two decades; however, the molecular machinery responsible for driving the system remains elusive. Thus, whether conserved PCD regulatory mechanisms include plant apoptosis remains enigmatic. Animal apoptotic regulators, including Bcl-2 family members, have not been identified in plants but expression of such regulators can trigger or suppress plant PCD. Moreover, plants exhibit nearly all of the biochemical and morphological features of apoptosis. One difference between plant and animal PCD is the absence of phagocytosis in plants. Evidence is emerging that the vacuole may be key to removal of unwanted plant cells, and may carry out functions that are analogous to animal phagocytosis. Here, we provide context for the argument that apoptotic-like cell death occurs in plants.

SummaryEndoplasmic reticulum (ER) stress due to biotic or abiotic stress activates the unfolded protein response (UPR) to restore ER homeostasis. The UPR relies on multiple ER-to-nucleus signaling factors which mainly induce the expression of cytoprotective ER-chaperones. The inositol requiring enzyme (IRE1) along with its splicing target, bZIP60, restrict potyvirus, and potexvirus accumulation. Until now, the involvement of the alternative UPR pathways and the role of UPR to limit virus accumulation have remained elusive. Here, we used the Plantago asiatica mosaic virus (PlAMV) and the Turnip mosaic virus (TuMV) to demonstrate that the potexvirus triple gene block 3 (TGB3) protein and the potyvirus 6K2 protein activate the bZIP17, bZIP28, bZIP60, BAG7, NAC089 and NAC103 signaling in Arabidopsis thaliana. Using the corresponding knock-out mutant lines, we demonstrated that these factors differentially restrict local and systemic virus accumulation. We show that bZIP17, bZIP60, BAG7, and NAC089 are factors in PlAMV infection, whereas bZIP28 and bZIP60 are factors in TuMV infection. TGB3 and 6K2 transient expression in leave reveal that these alternative pathways induce BiPs expression. Finally, using dithiothreitol (DTT) and tauroursodeoxycholic acid (TUDCA) treatment, we demonstrated that the protein folding capacity significantly influences PlAMV accumulation. Together, these results indicate that multiple ER-to-nucleus signaling pathways are activated during virus infection and restrict virus accumulation through increasing protein folding capacity.Significance statementThe IRE1/bZIP60 pathway of unfolded protein response (UPR) is activated by potyviruses and potexviruses, limiting their infection, but the role of alternative UPR pathways is unknown. This study reveals the activation of multiple ER-to-nucleus signaling pathways by the Plantago asiatica mosaic virus and the Turnip mosaic virus. We identify additional signaling pathways serve to restrict virus accumulation through increased protein folding capacity.

The Bcl-2 associated athanogene (BAG) family is an evolutionarily conserved group of co-chaperones that confers stress protection against a variety of cellular insults extending from yeasts, plants to humans. Little is known, however, regarding the biological role of BAG proteins in phytopathogenic fungi. Here, we identified the unique BAG gene (BcBAG1) from the necrotrophic fungal pathogen, Botrytis cinerea. BcBAG1 is the homolog of Arabidopsis thaliana AtBAG4, and ectopic expression of BcBAG1 in atbag4 knock-out mutants restores salt tolerance. BcBAG1 deletion mutants (ΔBcbag1) exhibited decreased conidiation, enhanced melanin accumulation and lost the ability to develop sclerotia. Also, BcBAG1 disruption blocked fungal conidial germination and successful penetration, leading to a reduced virulence in host plants. BcBAG1 contains BAG (BD) domain at C-terminus and ubiquitin-like (UBL) domain at N-terminus. Complementation assays indicated that BD can largely restored pathogenicity of ΔBcbag1. Abiotic stress assays showed ΔBcbag1 was more sensitive than the wild-type strain to NaCl, calcofluor white, SDS, tunicamycin, dithiothreitol (DTT), heat and cold stress, suggesting BcBAG1 plays a cytoprotective role during salt stress, cell wall stress, and ER stress. BcBAG1 negatively regulated the expression of BcBIP1, BcIRE1 and the splicing of BcHAC1 mRNA, which are core regulators of unfolded protein response (UPR) during ER stress. Moreover, BcBAG1 interacted with HSP70-type chaperones, BcBIP1 and BcSKS2. In summary, this work demonstrates that BcBAG1 is pleiotropic and not only essential for fungal development, hyphal melanization, and virulence, but also required for response to multiple abiotic stresses and UPR pathway of B. cinerea.

Plants and fungi have had many years of friendly and not-so friendly competition for resources and quality of life. As a result, diverse pathosystems evolved numerous strategies, coupled with the emergence of multifaceted pathogenic and saprophytic lifestyles. We discuss fungal lifestyle classifications and how the views associated with certain fungal pathogens, particularly necrotophs, are changing as we learn more about the complexities of their interactions with a given host plant. We discuss the physiological events leading to the transition from biotrophy to necrotrophy in hemi-biotrophs, and conclude that both the control of plant immune responses and the need for a more efficient mode of nutrient acquisition are possible triggers for the transition to necrotrophy. Based on recent findings, we focus on the polyphagous plant pathogen Sclerotinia sclerotiorum. Rather than overwhelming plant foes, S. sclerotiorum has evolved clever means to compromise host recognition and establish disease, resulting in a broad and immensely successful pathogenic lifestyle. The tactics used by this fungus to achieve pathogenic success are being clarified. We propose that the hemi-biotrophic lifestyle may be more temporally and spatially complex than currently depicted, and that combining lifestyle attributes with damage response curves that consider the contribution of both the fungus and the host to pathogenesis, may provide a more holistic manner to view plant pathogens.

The soilborne necrotrophic pathogens Rhizoctonia and Pythium infect a wide range of crops in the US and worldwide. These pathogens pose challenges to growers because the diseases they cause are not adequately controlled by fungicides, rotation or, for many hosts, natural genetic resistance. Although a combination of management practices are likely to be required for control of Rhizoctonia and Pythium, genetic resistance remains a key missing component. This review discusses the recent deployment of introduced genes and genome-based information for control of Rhizoctonia, with emphasis on three pathosystems: Rhizoctonia solani AG8 and wheat, R. solani AG1-IA and rice, and R. solani AG3 or AG4 and potato. Molecular mechanisms underlying disease suppression will be addressed, if appropriate. Although less is known about genes and factors suppressive to Pythium, pathogen genomics and biological control studies are providing useful leads to effectors and antifungal factors. Prospects for resistance to Rhizoctonia and Pythium spp. will continue to improve with growing knowledge of pathogenicity strategies, host defense gene action relative to the pathogen infection process, and the role of environmental factors on pathogen-host interactions.

DNA contains all the information needed to build a body, yet molecules of RNA carry these instructions to the sites in the cell where they can be used. Cells must control how much RNA they produce in order to ensure that they develop properly and can respond well to their environment. RNA silencing refers to a collection of mechanisms that use smaller RNA molecules called microRNAs to incapacitate certain RNA molecules and selectively switch off the genes that encode them to stop more from being made. One key player in RNA silencing is the multi-protein complex called RISC, which contains microRNA and a group of proteins called AGOs. Once the microRNA has identified its RNA target, the AGOs cut the RNA into two pieces, known as the 5’ cleavage fragment and 3’ cleavage fragment. The two resulting fragments need to be cleared away swiftly, so that the RISC can move on to the next target. While it was known how the 3’ cleavage fragment was removed, it was less clear how the 5’ cleavage fragment was dealt with. Previous studies had shown that the 5’ cleavage fragment was marked with a chemical called uridine, which somehow signals to the RISC that this fragment needs to be destroyed. Now, using biochemical techniques, Zhang et al. have identified two new proteins in the model plant Arabidopsis that attach to the AGO proteins and degrade the 5’ cleavage fragments that are marked with uridine. The two proteins are named RICE1 and RICE2. Zhang et al. then analyzed the three-dimensional shape of RICE1 and identified the ‘active’ region that is responsible for degrading the RNA fragments. When these active regions were blocked, the microRNA levels were low, but the uridine-marked 5’ cleavage fragments were high. Also, the RISC complex could not work properly, which lead to problems during the development of the plant. These results suggest that RICE proteins degrade 5’ cleavage fragments modified with uridine to activate RISC. RICE proteins are conserved between plants and animals, and it is likely that their counterparts in humans will have a similar role to the plant proteins. The next challenge will be to explore how RICE proteins work in more details, which may lead to new ways to manipulate the levels of microRNAs to change the architecture of the plant and to improve their tolerance to various stress conditions.

Reactive oxygen species (ROS) are essential for pathogenic development of Sclerotinia sclerotiorum . A key question for S. sclerotiorum and many other pathogens concerns how fungi tolerate/dampen the oxidative environment during growth and pathogenesis. Regulatory components of oxidative stress include both enzymatic and non-enzymatic antioxidants. Catalases are a ubiquitous family of enzymes that play an important role in the enzymatic detoxification of ROS by converting hydrogen peroxide (H 2 O 2 ) to water and molecular oxygen. The genome of the omnivorous pathogen S. sclerotiorum contains seven predicted catalase genes. In this study we evaluate and functionally characterize the type A catalase ( Scat1 ) in S. sclerotiorum , whose expression is highly induced during host infection. Insertional inactivation of Scat1 (Δ Scat1 ) resulted in hyperbranching of hyphae accompanied by slower growth and smaller sclerotia. Δ Scat1 strains were attenuated in pathogenicity and rendered the fungus hypersensitive to Sodium dodecyl sulfate (SDS), as well as to osmotic and salt stresses. Unexpectedly, Δ Scat1 exhibited increased tolerance to H 2 O 2 , suggesting that although a member of the catalase family, generally associated with amelioration of oxidative stress, Scat1 is probably not required for detoxification of this oxygen species and presumably has different function(s). Δ Scat1 strains had a 2-fold decrease in ergosterol content, and overall lower sterol levels compared to the wild-type strain. These observations are consistent with increased resistance to the polyene drugs amphotericin-B and nystatin. Taken together, our results suggest Scat1 is involved in modulation of ROS in a manner that deviates from the detoxification of H 2 O 2 , alters membrane integrity and contributes to the pathogenic success of S. sclerotiorum .

Programmed cell death (PCD) is essential for proper growth, development, and cellular homeostasis in all eukaryotes. The regulation of PCD is of central importance in plant-microbe interactions; notably, PCD and features associated with PCD are observed in many host resistance responses. Conversely, pathogen induction of inappropriate cell death in the host results in a susceptible phenotype and disease. Thus, the party in control of PCD has a distinct advantage in these battles. PCD processes appear to be of ancient origin, as indicated by the fact that many features of cell death strategy are conserved between animals and plants; however, some of the details of death execution differ. Mammalian core PCD genes, such as caspases, are not present in plant genomes. Similarly, pro- and antiapoptotic mammalian regulatory elements are absent in plants, but, remarkably, when expressed in plants, successfully impact plant PCD. Thus, subtle structural similarities independent of sequence homology appear to sustain operational equivalence. The vacuole is emerging as a key organelle in the modulation of plant PCD. Under different signals for cell death, the vacuole either fuses with the plasmalemma membrane or disintegrates. Moreover, the vacuole appears to play a key role in autophagy; evidence suggests a prosurvival function for autophagy, but other studies propose a prodeath phenotype. Here, we describe and discuss what we know and what we do not know about various PCD pathways and how the host integrates signals to activate salicylic acid and reactive oxygen pathways that orchestrate cell death. We suggest that it is not cell death as such but rather the processes leading to cell death that contribute to the outcome of a given plant-pathogen interaction.

The Potato virus X (PVX) triple gene block protein 3 (TGBp3), an 8-kDa membrane binding protein, aids virus movement and induces the unfolded protein response (UPR) during PVX infection. TGBp3 was expressed from the Tobacco mosaic virus (TMV) genome (TMV-p3), and we noted the up-regulation of SKP1 and several endoplasmic reticulum (ER)-resident chaperones, including the ER luminal binding protein (BiP), protein disulphide isomerase (PDI), calreticulin (CRT) and calmodulin (CAM). Local lesions were seen on leaves inoculated with TMV-p3, but not TMV or PVX. Such lesions were the result of TGBp3-elicited programmed cell death (PCD), as shown by an increase in reactive oxygen species, DNA fragmentation and induction of SKP1 expression. UPR-related gene expression occurred within 8 h of TMV-p3 inoculation and declined before the onset of PCD. TGBp3-mediated cell death was suppressed in plants that overexpressed BiP, indicating that UPR induction by TGBp3 is a pro-survival mechanism. Anti-apoptotic genes Bcl-xl, CED-9 and Op-IAP were expressed in transgenic plants and suppressed N gene-mediated resistance to TMV, but failed to alleviate TGBp3-induced PCD. However, TGBp3-mediated cell death was reduced in SKP1-silenced Nicotiana benthamiana plants. The combined data suggest that TGBp3 triggers the UPR and elicits PCD in plants.

To cope with stress and increased accumulation of misfolded proteins, plants and animals use a survival pathway known as the unfolded protein response (UPR) that signals between the endoplasmic reticulum (ER) and the nucleus to maintain cell homeostasis via proper folding of proteins. B-cell lymphoma2 (Bcl-2)-associated athanogene (BAG) proteins are an evolutionarily conserved family of co-chaperones that are linked to disease states in mammals and responses to environmental stimuli (biotic and abiotic) in plants. Molecular and physiological techniques were used to functionally characterize a newly identified branch of the UPR initiated by the ER-localized co-chaperone from Arabidopsis thaliana, AtBAG7. AtBAG7 has functional roles in both the ER and the nucleus. Upon heat stress, AtBAG7 is sumoylated, proteolytically processed and translocated from the ER to the nucleus, where interaction with the WRKY29 transcription factor occurs. Sumoylation and translocation are required for the AtBAG7–WRKY29 interaction and subsequent stress tolerance. In the ER, AtBAG7 interacts with the ER-localized transcription factor, AtbZIP28, and established UPR regulator, the AtBiP2 chaperone. The results indicate that AtBAG7 plays a central regulatory role in the heat-induced UPR pathway.

One of the earliest plant responses to pathogens is the induced accumulation of reactive oxygen species (ROS). The superoxide ion is an important intermediate in the generation of ROS having a key regulatory function during plant–microbe interactions and is an important component in fungal development. The superoxide dismutase (SOD) family contributes to frontline defense via detoxification of reactive superoxide radical anions. Sclerotinia sclerotiorum (Lib.) de Bary is a necrotrophic fungal pathogen with a broad host range. S. sclerotiorum produces the non-specific phytotoxin and key pathogenicity factor, oxalic acid (OA). We have identified an S. sclerotiorum SOD (Sssod1) with high similarity to CuZnSODs. Sssod1 contains an open reading frame of 908 bp in length and is predicted to encode a protein of 155 amino acids that harbors the entire hallmark motifs associated with SOD function. Treatment with the CuZnSOD inhibitor diethyldithiocarbamate (DETC) resulted in delayed hyphal growth and sclerotial development in a dose-dependent manner. Mutants generated carrying an Sssod1 deletion (ΔSssod1) exhibited morphological defects similar to those observed with the inhibitor treatment. Moreover, ΔSssod1 was more sensitive than wild-type to menadione, a redox cycling agent. Expression of Sssod1 was induced following treatment with oxidizing agents and during interaction with plant host tissue the ΔSssod1 mutant was significantly reduced in virulence on both tomato and tobacco plants compared to wild-type. Interestingly, pathogenicity of the superoxide dismutase mutant was mostly restored following supplementation with oxalate. We also observed that ΔSssod1 was reduced in oxalate production by half. In accordance with reduced virulence, ΔSssod1 induced a host oxidative burst in adjacent uninfected cells, a phenotype indicative of active pathogen recognition by the host. Intriguingly, during wild-type infection, host ROS production was significantly reduced. These results suggest that wild-type Sclerotinia suppresses host defense responses during infection.

Abscission occurs specifically in the abscission zone (AZ) tissue as a natural stage of plant development. Previously, we observed delay of tomato (Solanum lycopersicum) leaf abscission when the LX ribonuclease (LX) was inhibited. The known association between LX expression and programmed cell death (PCD) suggested involvement of PCD in abscission. In this study, hallmarks of PCD were identified in the tomato leaf and flower AZs during the late stage of abscission. These included loss of cell viability, altered nuclear morphology, DNA fragmentation, elevated levels of reactive oxygen species and enzymatic activities, and expression of PCD-associated genes. Overexpression of antiapoptotic proteins resulted in retarded abscission, indicating PCD requirement. PCD, LX, and nuclease gene expression were visualized primarily in the AZ distal tissue, demonstrating an asymmetry between the two AZ sides. Asymmetric expression was observed for genes associated with cell wall hydrolysis, leading to AZ, or associated with ethylene biosynthesis, which induces abscission. These results suggest that different abscission-related processes occur asymmetrically between the AZ proximal and distal sides. Taken together, our findings identify PCD as a key mechanism that occurs asymmetrically during normal progression of abscission and suggest an important role for LX in this PCD process.

Numerous studies have shown both the detrimental and beneficial effects of reactive oxygen species (ROS) in animals, plants, and fungi. These organisms utilize controlled generation of ROS for signaling, pathogenicity, and development. Here, we show that ROS are essential for the pathogenic development of Sclerotinia sclerotiorum , an economically important fungal pathogen with a broad host range. Based on the organism's completed genome sequence, we identified two S. sclerotiorum NADPH oxidases (SsNox1 and SsNox2), which presumably are involved in ROS generation. RNA interference (RNAi) was used to examine the function of SsNox1 and SsNox2. Silencing of SsNox1 expression indicated a central role for this enzyme in both virulence and pathogenic (sclerotial) development, while inactivation of the SsNox2 gene resulted in limited sclerotial development, but the organism remained fully pathogenic. Δ Ssnox1 strains had reduced ROS levels, were unable to develop sclerotia, and unexpectedly correlated with significantly reduced oxalate production. These results are in accordance with previous observations indicating that fungal NADPH oxidases are required for pathogenic development and are consistent with the importance of ROS regulation in the successful pathogenesis of S. sclerotiorum .

Fusarium wilt, caused by Fusarium oxysporum f. sp. cubense (Foc), is one of the most devastating diseases of banana (Musa spp.). Apart from resistant cultivars, there are no effective control measures for the disease. We investigated whether the transgenic expression of apoptosis-inhibition-related genes in banana could be used to confer disease resistance. Embryogenic cell suspensions of the banana cultivar, 'Lady Finger', were stably transformed with animal genes that negatively regulate apoptosis, namely Bcl-xL, Ced-9 and Bcl-2 3' UTR, and independently transformed plant lines were regenerated for testing. Following a 12-week exposure to Foc race 1 in small-plant glasshouse bioassays, seven transgenic lines (2 × Bcl-xL, 3 × Ced-9 and 2 × Bcl-2 3' UTR) showed significantly less internal and external disease symptoms than the wild-type susceptible 'Lady Finger' banana plants used as positive controls. Of these, one Bcl-2 3' UTR line showed resistance that was equivalent to that of wild-type Cavendish bananas that were included as resistant negative controls. Further, the resistance of this line continued for 23-week postinoculation at which time the experiment was terminated. Using TUNEL assays, Foc race 1 was shown to induce apoptosis-like features in the roots of wild-type 'Lady Finger' plants consistent with a necrotrophic phase in the life cycle of this pathogen. This was further supported by the observed reduction in these effects in the roots of the resistant Bcl-2 3' UTR-transgenic line. This is the first report on the generation of transgenic banana plants with resistance to Fusarium wilt.

Previously, we showed that transgenic plants expressing SfIAP, an insect anti-apoptotic gene, conferred tolerance to several abiotic stresses and resistance to the fungal pathogen Alternaria alternata . A pronounced and consistent delay in tomato fruit ripening was also observed. Preliminary data suggested that SfIAP negatively affected ethylene synthesis, which could account, at least in part, for the observed phenotypes. A key question is how an insect anti-apoptotic protein functions to enhance stress tolerance in plants, particularly since conserved core apoptotic pathway regulators appear to be absent in plants at the primary sequence level. In this study, we show that the RING domain, a characteristic motif of several IAP family members, has an E3 ubiquitin ligase activity suggesting involvement of SfIAP in proteasome-mediated protein degradation. Proteasome inhibitors were applied to transgenic plants undergoing stress. Proteasome inhibitor treatment restored ethylene signaling and wild type phenotypes in SfIAP expressing transgenic plants. Transient assays of RING domain deletion (Sf∆RING) or point mutations (SfIAPC330A) resulted in the inhibition of ubiquitin ligase activity, while also restoring normal ethylene signaling and the wild type phenotypes including pathogen susceptibility and normal fruit ripening in tomato. During salt stress, increases in ubiquitinylated proteins correlated with salt tolerance in SfIAP expressing plants. Baculovirus inverted repeat (BIR) domains, signature motifs of the IAP family, are also required for the full resistant phenotype of SfIAP plants. SfBIR1, but not SFBIR2, is required for salt tolerance. Only when coupled with the RING domain, was protection conferred in a similar manner to full length SfIAP against salt stress. Taken together, the ubiquitin ligase activity of SfIAP is necessary, but not sufficient to confer plant stress tolerance. The mechanism/target by which SfIAP confers tolerance appears to be via ethylene regulation and the ubiquitin/proteasome pathway.

SfIAP, an IAP ( i nhibitor of apoptosis) family member from the insect Spodoptera frugiperda , was introduced into tobacco and tomato plants. Expression of SfIAP conferred tolerance to several abiotic stresses including heat, salt, and fumonisin B1 (FB1) as well as resistance to the necrotrophic fungus Alternaria alternata . In wild type tomato, these stresses induced DNA fragmentation and formation of apoptotic-like bodies prior to cell death. Transgenic expression of SfIAP inhibited these features and cell death did not occur. In addition, tomato fruit ripening was delayed in plants harboring SfIAP. Expression of EIN3, a key transcription factor mediating ethylene responses, was suppressed in transgenic plants during fruit ripening and in response to fungal challenge. Treatment of transgenic plants with ethephon restored normal fruit ripening and disease susceptibility. The expression of ethylene-associated genes during the process of fruit ripening was blocked in SfIAP-expressing plants. The phenotypes associated with expression of SfIAP were not correlated with the expression of pathogenesis-related proteins. Taken together, these data suggest that the insect anti-apoptotic gene SfIAP, has a conserved function that inhibits stress-induced cell death both in plants and animals.

The BAG (Bcl-2 associated athanogene) family is a multifunctional group of proteins that perform diverse functions ranging from apoptosis to tumorigenesis. An evolutionarily conserved group, these proteins are distinguished by a common conserved region known as the BAG domain. BAG genes have been found in yeasts, plants, and animals, and are believed to function as adapter proteins forming complexes with signaling molecules and molecular chaperones. In humans, a role for BAG proteins has been suggested in carcinogenesis, HIV infection, and Parkinson's disease. These proteins are therefore potential therapeutic targets, and their expression in cells may serve as a predictive tool for such diseases. In plants, the Arabidopsis thaliana genome contains seven homologs of the BAG family, including four with domain organization similar to animal BAGs. Three members contain a calmodulin-binding domain possibly reflecting differences between plant and animal programmed cell death. This review summarizes current understanding of BAG proteins in both animals and plants.

Sclerotia are hard, asexual, resting structures which can survive for years in soil. In Sclerotinia sclerotiorum, which provides a good model system for studying sclerotial development, sclerotial development has been traditionally divided to three macroscopically distinct stages (initiation, development and maturation). However, additional phases (which can be visualized microscopically) indicate a complex, multi-step, process is involved. Environmental changes, primary metabolism and secondary messengers have been well documented factors affecting sclerotial development, yet analysis of the molecular mechanisms involved in sclerotial development is in its infancy. Here, we review the current status of the known molecular components involved in sclerotial development, with an emphasis on phosphorylative regulation of sclerotial development in S. sclerotiorum . Components such as cAMP-dependent protein kinase, ERK-like mitogen-activated protein kinase and Ser/Thr phosphatases type 2A and 2B, shown to regulate other developmental processes in fungi, have recently been shown to also be involved in regulation of sclerotium development. Reversible protein phosphorylation, as well as additional regulatory mechanisms of gene expression such as DNA methylation and ribosome inactivation, most likely function in concert with secondary metabolites, reactive oxygen species, pH and light in order to regulate sclerotial development in different fungi. The diversity of sclerotium-producing fungi promises to yield exciting variations into the molecular mechanisms regulating this developmental process in different species.

The death of plant cells in culture following exposure to Agrobacterium tumefaciens remains a major obstacle in developing Agrobacterium-mediated transformation into a highly efficient genotype-independent technology. Here, we present evidence that A. tumefaciens exposure induces cell death in banana cell suspensions. More than 90% of embryogenic banana cells died after exposure to A. tumefaciens and cell death was accompanied by a subset of features associated with apoptosis in mammalian cells, including DNA laddering, fragmentation, and formation of apoptoticlike bodies. Importantly, these cellular responses were inhibited in cells expressing the animal antiapoptosis genes Bcl-xL, Bcl-2 3′ untranslated region, and CED-9. Inhibition of cell death resulted in up to 90% of cell clumps transformed with Bcl-xL, a 100-fold enhancement over vector controls, approaching the transformation and regeneration of every “transformable” cell. Similar results using sugarcane, a crop plant known for recalcitrance to Agrobacterium transformation, suggest that antiapoptosis genes may inhibit these phenomena and increase the transformation frequency of many recalcitrant plant species, including the major monocot cereal crop plants. Evidence of inhibition of plant cell death by cross-kingdom antiapoptotic genes also contributes to the growing evidence that genes for control of programmed cell death are conserved across wide evolutionary distances, even though these mechanisms are not well understood in plants. Additional keywords: necrosis.

The long-term goals of our research are to understand the regulation of sclerotial development and pathogenicity in S. sclerotior11111. The focus in this project was on the elucidation of the signaling events and environmental cues involved in the regulation of these processes, utilizing and continuously developing tools our research groups have established and/or adapted for analysis of S. sclerotiorum, Our stated objectives: To take advantage of the recent conceptual (ROS/PPs signaling) and technical (amenability of S. sclerotiorumto manipulations coupled with chemical genomics and next generation sequencing) developments to address and extend our fundamental and potentially applicable knowledge of the following questions concerning the involvement of REDOX signaling and protein dephosphorylation in the regulation of hyphal/sclerotial development and pathogenicity of S. sclerotiorum: (i) How do defects in genes involved in ROS signaling affect S. sclerotiorumdevelopment and pathogenicity? (ii) In what manner do phosphotyrosinephosphatases affect S. sclerotiorumdevelopment and pathogenicity and how are they linked with ROS and other signaling pathways? And (iii) What is the nature of activity of newly identified compounds that affect S. sclerotiori,111 growth? What are the fungal targets and do they interfere with ROS signaling? We have met a significant portion of the specific goals set in our research project. Much of our work has been published. Briefly. we can summarize that: (a) Silencing of SsNox1(NADPHoxidase) expression indicated a central role for this enzyme in both virulence and pathogenic development, while inactivation of the SsNox2 gene resulted in limited sclerotial development, but the organism remained fully pathogenic. (b) A catalase gene (Scatl), whose expression was highly induced during host infection is involved in hyphal growth, branching, sclerotia formation and infection. (c) Protein tyrosine phosphatase l (ptpl) is required for sclerotial development and is involved in fungal infection. (d) Deletion of a superoxidedismutase gene (Sssodl) significantly reduced in virulence on both tomato and tobacco plants yet pathogenicity was mostly restored following supplementation with oxalate. (e) We have participated in comparative genome sequence analysis of S. sclerotiorumand B. cinerea. (f) S. sclerotiorumexhibits a potential switch between biotrophic and necrotrophic lifestyles (g) During plant­ microbe interactions cell death can occur in both resistant and susceptible events. Non­ pathogenic fungal mutants S. sclerotior111n also cause a cell death but with opposing results. We investigated PCD in more detail and showed that, although PCD occurs in both circumstances they exhibit distinctly different features. The mutants trigger a restricted cell death phenotype in the host that unexpectedly exhibits markers associated with the plant hypersensitive (resistant) response. Using electron and fluorescence microscopy, chemical effectors and reverse genetics, we have established 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 interaction Thus the control of cell death, dictated by the plant (autophagy) סr the fungus (apoptosis), is decisive to the outcome of certain plant­ microbe interactions. In addition to the time and efforts invested towards reaching the specific goals mentioned, both Pls have initiated utilizing (as stated as an objective in our proposal) state of the art RNA-seq tools in order to harness this technology for the study of S. sclerotiorum. The Pls have met twice (in Israel and in the US), in order to discuss .נחd coordinate the research efforts. This included a working visit at the US Pls laboratory for performing RNA-seq experiments and data analysis as well as working on a joint publication (now published). The work we have performed expands our understanding of the fundamental biology (developmental and pathogenic) of S. sclerotioז111וז. Furthermore, based on our results we have now reached the conclusion that this fungus is not a bona fide necrotroph, but can also display a biotrophic lifestyle at the early phases of infection. The data obtained can eventually serve .נ basis of rational intervention with the disease cycle of this pathogen.

Global climate change, increasingly erratic weather and a burgeoning global population are significant threats to the sustainability of future crop production. There is an urgent need for the development of robust measures that enable crops to withstand the uncertainty of climate change whilst still producing maximum yields. Resurrection plants possess the unique ability to withstand desiccation for prolonged periods, can be restored upon watering and represent great potential for the development of stress tolerant crops. Here, we describe the remarkable stress characteristics of Tripogon loliiformis, an uncharacterised resurrection grass and close relative of the economically important cereals, rice, sorghum, and maize. We show that T. loliiformis survives extreme environmental stress by implementing autophagy to prevent Programmed Cell Death. Notably, we identified a novel role for trehalose in the regulation of autophagy in T.loliiformis. Transcriptome, Gas Chromatography Mass Spectrometry, immunoblotting and confocal microscopy analyses directly linked the accumulation of trehalose with the onset of autophagy in dehydrating and desiccated T. loliiformis shoots. These results were supported in vitro with the observation of autophagosomes in trehalose treated T. loliiformis leaves; autophagosomes were not detected in untreated samples. Presumably, once induced, autophagy promotes desiccation tolerance in T.loliiformis, by removal of cellular toxins to suppress programmed cell death and the recycling of nutrients to delay the onset of senescence. These findings illustrate how resurrection plants manipulate sugar metabolism to promote desiccation tolerance and may provide candidate genes that are potentially useful for the development of stress tolerant crops., Author Summary Over coming decades, climate change models suggest that droughts and other unpredictable weather patterns will appear more frequently. It is imperative that we develop crops that can survive future climates but continue to yield. Numerous studies have shown that stress tolerance is genetically encoded. Naturally tolerant species therefore represent an ideal starting point for the search for stress tolerance. Resurrection plants belong to a small group of vascular plants that possess unique stress tolerance mechanisms to withstand extreme desiccation with the ability to recover fully upon the availability of water. Here we describe a unique regulatory role for trehalose in the activation of autophagy pathways in T.loliiformis. We show that T. loliiformis leaves are alive in desiccated plants and that pre-existing tissues resurrect upon the addition of water. By using a combination of transcriptomics, confocal microscopy and spectroscopy we show that autophagy is induced during dehydration. Notably, we establish that treatment of leaves with trehalose triggers autophagy in vitro and that trehalose accumulation in dehydrating leaves correlates with the presence of autophagosomes. We postulate that resurrection plants modulates trehalose metabolism to induce and maintain autophagy pathways that preventing senescence and programmed cell death.

Sclerotinia sclerotiorum is a filamentous fungus (mold) that causes plant disease. It has an extremely wide range of hosts (>400 species) and causes considerable damage (annual multimillion dollar losses) in economically important crops. It has proven difficult to control (culturally or chemically) and host resistance to this fungus has generally been inadequate. It is believed that this fungus occurs in almost every country. Virulence of this aggressive pathogen is bolstered by a wide array of plant cell wall degrading enzymes and various compounds (secondary metabolites) produced by the fungus. It is well established that plant pathogenic fungi secrete proteins and small molecules that interact with host cells and play a critical role in disease development. Such secreted proteins have been collectively designated as “effectors”. Plant resistance against some pathogens can be mediated by recognition of such effectors. Alternatively, effectors can interfere with plant defense. Some such effectors are recognized by the host plant and can culminate in a programmed cell death (PCD) resistant response. During the course of this study, we analyzed an effector in Sclerotiniasclerotiorum. This specific effector, SsCM1 is the protein chorismatemutase, which is an enzyme involved in a pathway which is important in the production of important amino acids, such a Tryptophan. We have characterized the Sclerotiniaeffector, SsCM1, and have shown that inactivation of Sscm1 does not affect fungal vegetative growth, development or production of oxalic acid (one of this fungus’ secondary metabolites associated with disease) production. However, yhis does result in reduced fungal virulence. We show that, unexpectedly, the SsCM1 protein translocates to the host chloroplast, and demonstrated that this process is required for full fungal virulence. We have also determined that the fungal SsCM1 protein can interact with similar proteins produced by the host. In addition, we have shown that the fungal SsCM1 is able to suppress at least some of the effects imposed by reactive oxygen species which are produced as a defense mechanism by the host. Last, but not least, the results of our studies have provided evidence contradicting the current dogma on at least some of the mechanist aspects of how this pathogen infects the host. Contrary to previousons, indicating that this pathogen kills its host by use of metabolites and enzymes that degrade the host tissue (a process called necrotrophy), we now know that at least in the early phases of infection, the fungus interacts with live host tissue (a phenomenon known as biotrophy). Taken together, the results of our studies provide novel insights concerning the mechanistic aspects of Sclerotinia-host interactions. We hope this information will be used to interfere with the disease cycle in a manner that will protect plants from this devastating fungus.

Abstract Recent advances in sequencing technologies have led to a remarkable increase in the number of sequenced fungal genomes. Several important plant pathogenic fungi are among those that have been sequenced or are being sequenced. Additional fungal pathogens are likely to be sequenced in the near future. Analysis of the available genomes has provided useful information about genes that may be important for plant infection and colonization. Genome features, such as repetitive sequences, telomeres, conserved syntenic blocks, and expansion of pathogenicity-related genes, are discussed in detail with Magnaporthe oryzae (M. grisea) and Fusarium graminearum as examples. Functional and comparative genomic studies in plant pathogenic fungi, although still in the early stages and limited to a few pathogens, have enormous potential to improve our understanding of the molecular mechanisms involved in host-pathogen interactions. Development of advanced genomics tools and infrastructure is critical for efficient utilization of the vast wealth of available genome sequence information and will form a solid foundation for systems biology studies of plant pathogenic fungi.

The genes that control mammalian programmed cell death are conserved across wide evolutionary distances. Although plant cells can undergo apoptosis-like cell death, plant homologs of mammalian regulators of apoptosis have, in general, not been found. This is in part due to the lack of primary sequence conservation between animal and putative plant regulators of apoptosis. Thus, alternative approaches beyond sequence similarities are required to find functional plant homologs of apoptosis regulators. Here, we present the results of using advanced bioinformatic tools to uncover the Arabidopsis family of BAG proteins. The mammalian BAG (Bcl-2-associated athanogene) proteins are a family of chaperone regulators that modulate a number of diverse processes ranging from proliferation to growth arrest and cell death. Such proteins are distinguished by a conserved BAG domain that directly interacts with Hsp70 and Hsc70 proteins to regulate their activity. Our searches of the Arabidopsis thaliana genome sequence revealed seven homologs of the BAG protein family. We further show that plant BAG family members are also multifunctional and remarkably similar to their animal counterparts, as they regulate apoptosis-like processes ranging from pathogen attack to abiotic stress and development.

Exogenous proline can protect cells of Saccharomyces cerevisiae from oxidative stress. We altered intracellular proline levels by overexpressing the proline dehydrogenase gene ( PUT1 ) of S. cerevisiae . Put1p performs the first enzymatic step of proline degradation in S. cerevisiae . Overexpression of Put1p results in low proline levels and hypersensitivity to oxidants, such as hydrogen peroxide and paraquat. A put1 -disrupted yeast mutant deficient in Put1p activity has increased protection from oxidative stress and increased proline levels. Following a conditional life/death screen in yeast, we identified a tomato ( Lycopersicon esculentum ) gene encoding a QM-like protein (tQM) and found that stable expression of tQM in the Put1p-overexpressing strain conferred protection against oxidative damage from H 2 O 2 , paraquat, and heat. This protection was correlated with reactive oxygen species (ROS) reduction and increased proline accumulation. A yeast two-hybrid system assay was used to show that tQM physically interacts with Put1p in yeast, suggesting that tQM is directly involved in modulating proline levels. tQM also can rescue yeast from the lethality mediated by the mammalian proapoptotic protein Bax, through the inhibition of ROS generation. Our results suggest that tQM is a component of various stress response pathways and may function in proline-mediated stress tolerance in plants.

Cdc42 is a highly conserved small GTP-binding protein that is involved in regulating morphogenesis in eukaryotes. In this study, we isolated and characterized a highly conserved Cdc42 gene from Colletotrichum trifolii (Ct Cdc42 ), a fungal pathogen of alfalfa. CtCdc42 is, at least in part, functionally equivalent to Saccharomyces cerevisiae Cdc42p, since it restores the temperature-sensitive phenotype of a yeast Cdc42p mutant. Inhibition of CtCdc42 by expression of an antisense Ct Cdc42 or a dominant negative form of CtCdc42 (DN Cdc42) resulted in appressorium differentiation under noninductive conditions, suggesting that CtCdc42 negatively regulates pathogenic development in this fungus. We also examined the possible linkage between CtCdc42 and Ras signaling. Expression of a dominant active Cdc42 (DA Cdc42) in C. trifolii leads to aberrant hyphal growth under nutrient-limiting conditions. This phenotype was similar to that of our previously reported dominant active Ras (DA Ras) mutant. Also consistent with our observations of the DA Ras mutant, high levels of reactive oxygen species (ROS) were observed in the DA Cdc42 mutant, and proline restored the wild-type phenotype. Moreover, overexpression of DN Cdc42 resulted in a significant decrease in spore germination, virtually no hyphal branching, and earlier sporulation, again similar to what we observed in a dominant negative Ras (DN Ras) mutant strain. Interestingly, coexpression of DA Cdc42 with DN Ras resulted in germination rates close to wild-type levels, while coexpression of DN Cdc42 with the DA Ras mutant restored the wild-type phenotype. These data suggest that CtCdc42 is positioned as a downstream effector of CtRas to regulate spore germination and pathogenic development.

Sclerotinia sclerotiorum is a filamentous ascomycete phytopathogen able to infect an extremely wide range of cultivated plants. Our previous studies have shown that increases in cAMP levels result in the impairment of the development of the sclerotium, a highly differentiated structure important in the disease cycle of this fungus. cAMP also inhibits the activation of a S. sclerotiorum mitogen-activated protein kinase (MAPK), which we have previously shown to be required for sclerotial maturation; thus cAMP-mediated sclerotial inhibition is modulated through MAPK. However, the mechanism(s) by which cAMP inhibits MAPK remains unclear. Here we demonstrate that a protein kinase A (PKA)-independent signalling pathway probably mediates MAPK inhibition by cAMP. Expression of a dominant negative form of Ras, an upstream activator of the MAPK pathway, also inhibited sclerotial development and MAPK activation, suggesting that a conserved Ras/MAPK pathway is required for sclerotial development. Evidence from bacterial toxins that specifically inhibit the activity of small GTPases, suggested that Rap-1 or Ras is involved in cAMP action. The Rap-1 inhibitor, GGTI-298, restored MAPK activation in the presence of cAMP, further suggesting that Rap-1 is responsible for cAMP-dependent MAPK inhibition. Importantly, inhibition of Rap-1 is able to restore sclerotial development blocked by cAMP. Our results suggest a novel mechanism involving the requirement of Ras/MAPK pathway for sclerotial development that is negatively regulated by a PKA-independent cAMP signalling pathway. Cross-talk between these two pathways is mediated by Rap-1.

In mammalian cells, apoptosis is often mediated via organelles. While apoptotic-like cell death occurs in plants, the mechanistic details are unresolved. Transgenic tobacco plants have been generated that harbour selected animal anti-apoptotic genes. Subcellular fractionation followed by western blot analysis indicated that chloroplasts serve as a location for these animal anti-apoptotic proteins in addition to the established mitochondrial location. To explore the functional significance of this observation, tobacco plants were treated with three chloroplast-directed herbicides. Wild-type plants died and exhibited features associated with apoptosis. Transgenic plants survived and did not show any apoptotic-like characteristics. Moreover, the herbicide-induced apoptotic-like cell death was light requiring. It was concluded that chloroplasts may be involved in mediating certain types of plant programmed cell death.

Sclerotial development is fundamental to the disease cycle of the omnivorous broad host range fungal phytopathogen Sclerotinia sclerotiorum. We have isolated a highly conserved homolog of ERK-type mitogen-activated protein kinases (MAPKs) from S. sclerotiorum (Smk1) and have demonstrated that Smk1 is required for sclerotial development. The smk1 transcription and MAPK enzyme activity are induced dramatically during sclerotiogenesis, especially during the production of sclerotial initials. When PD98059 (a specific inhibitor of the activation of MAPK by MAPK kinase) was applied to differentiating cultures or when antisense expression of smk1 was induced, sclerotial maturation was impaired. The smk1 transcript levels were highest under acidic pH conditions, suggesting that Smk1 regulates sclerotial development via a pH-dependent signaling pathway, involving the accumulation of oxalic acid, a previously identified pathogenicity factor that functions at least in part by reducing pH. Addition of cyclic AMP (cAMP) inhibited smk1 transcription, MAPK activation, and sclerotial development. Thus, S. sclerotiorum can coordinate environmental signals (such as pH) to trigger a signaling pathway mediated by Smk1 to induce sclerotia formation, and this pathway is negatively regulated by cAMP.

Increased cAMP levels inhibit sclerotial development and raise oxalate production in Sclerotinia sclerotiorum. These effects were hypothesized to be mediated by protein kinase A (PKA). To test this hypothesis, a pka1 catalytic subunit gene was cloned and disrupted. Southern hybridization confirmed disruption, and northern analysis revealed loss of a wild-type pka1 transcript. Mutant strains were cAMP-responsive, pathogenic, and had wild-type levels of PKA activity. Phylogenetic analyses of pka1 with other fungal pka genes and encoded peptides suggest that filamentous fungi possess two pka paralogs. Multiple lines of evidence presented in this work suggest that a second pka catalytic subunit gene, pka2, exists and contributes the majority of PKA activity in S. sclerotiorum.

The small G-protein superfamily is an evolutionarily conserved group of GTPases that regulate diverse signalling pathways including pathways for growth and development in eukaryotes. Previously, we showed that dominant active mutation in the unique Ras gene (DARas) of the fungal phytopathogen Colletotrichum trifolii displays a nutrient-dependent phenotype affecting polarity, growth and differentiation. Signalling via the MAP kinase pathway is significantly impaired in this mutant as well. Here we describe the cloning and functional characterization of Rac (Ct-Rac1), a member of the Rho family of G proteins. Ct-Rac1 expression is downregulated by DARas under limiting nutrition. Co-expression of DARas with dominant active Rac (DARac) stimulates MAPK activation and restores the wild-type phenotype. Inhibition of MAPK activation suppresses phenotypic restoration suggesting Rac-mediated MAPK activation is responsible for reversion to the wild-type phenotype. We also examined the role of reactive oxygen species (ROS) in these genetic backgrounds. The DARas mutant strain generates high levels of ROS as determined by DCFH-DA fluorescence. Co-expression with DNRac decreases ROS generation to wild-type levels and restores normal fungal growth and development. Pretreatment of DARas with antioxidants or a cytosolic phospholipase A2 inhibitor also restores the wild-type phenotype. These findings suggest that Ras-mediated ROS generation is dependent on a Rac-cPLA 2 -linked signalling pathway. Taken together, this study provides evidence that Rac functions to restore the hyphal morphology of DARas by regulating MAPK activation and intracellular ROS generation.

Summary The Pseudomonas syringae pv. tomato DC3000 type III secretion system (TTSS) is required for bacterial pathogenicity on plants and elicitation of the hypersensitive response (HR), a programmed cell death (PCD) that occurs on resistant plants. Cosmid pHIR11 enables non-pathogens to elicit an HR dependent upon the TTSS and the effector HopPsyA. We used pHIR11 to determine that effectors HopPtoE, avirulence AvrPphEPto, AvrPpiB1Pto, AvrPtoB, and HopPtoF could suppress a HopPsyA-dependent HR on tobacco and Arabidopsis. Mixed inoculum and Agrobacterium-mediated transient expression experiments confirmed that suppressor action occurred within plant cells. These suppressors, with the exception of AvrPpiB1Pto, inhibited the expression of the tobacco pathogenesis-related (PR) gene PR1a. DC3000 suppressor mutants elicited an enhanced HR consistent with these mutants lacking an HR suppressor. Additionally, HopPtoG was identified as a suppressor on the basis of an enhanced HR produced by a hopPtoG mutant. Remarkably, these proteins functioned to inhibit the ability of the pro-apoptotic protein, Bax to induce PCD in plants and yeast, indicating that these effectors function as anti-PCD proteins in a trans-kingdom manner. The high proportion of effectors that suppress PCD suggests that suppressing plant immunity is one of the primary roles for DC3000 effectors and a central requirement for P. syringae pathogenesis.

Nicotiana tabacum L. ‘Glurk’ plants were transformed with antiapoptotic animal genes [chicken Bcl-xl; nematode CED-9; chicken Bcl-xl(GA) a mutant of Bcl-xl ; and a 3’ non-coding region of human Bcl-2, referred to as 161-1]. Our objectives were to determine whether plant transformation with anti-apoptotic genes ameliorates drought tolerance in tobacco plants by subjecting the plants to a dry-down period. The non-transformed Glurk and the transgenic Glurk harboring G115, which expresses β-glucuronidase, served as controls. Transformation of tobacco plants with animal anti-apoptotic genes significantly impacted the rates of photosynthesis (A) and stomatal conductance (gs), but not to the same extent in every line. Controls generally exhibited higher A and gs than the transformed plants under well-watered conditions. Photosynthesis and stomatal conductance declined significantly on the 3rd day, and approached zero on the 11th day of water deprivation. Differences among controls and transformed tobacco plants disappeared as soil moisture deficit increased. Relative water content (RWC) and leaf water potential (ψw) remained relatively high in the first 3 d of water deprivation, while a dramatic reduction occurred in all plants on the 6th day. Relative water content did not differ between controls and transformed plants. Water potential declined significantly (became more negative) with the increase in soil moisture deficit. Evident differences among transformed and control plants appeared on the 6th day of water deprivation. The controls, Glurk and G115 generally maintained a higher water potential between days 6 and 11 compared to the transformed tobacco plants. Osmotic adjustment occurred in transformed plants but not in the controls, in response to drought. Relative water content at turgor loss point (RWCtlp) and osmotic potential at turgor loss point (ψπtlp) provided a measure of drought tolerance in plants. G115, Glurk and Bcl-xl plants lost turgor at a higher relative water content than Bcl-xl(GA), CED-9 and 161-1. ψπtlp in the controls G115 and Glurk were relatively higher compared to the transformed plants. We concluded that transgenic plants with anti-apoptotic genes resulted in moderate amelioration of drought tolerance in tobacco plants. Key words: Anti-apoptotic genes, programmed cell death, genetic engineering, Nicotiana tabaccum, drought tolerance, gas exchange, photosynthesis, turgor, osmotic adjustment

When certain phytopathogenic fungi contact plant surfaces, specialized infection structures (appressoria) are produced that facilitate penetration of the plant external barrier; the cuticle. Recognition of this hydrophobic host surface must be sensed by the fungus, initiating the appropriate signaling pathway or pathways for pathogenic development. Using polymerase chain reaction and primers designed from mammalian protein kinase C sequences (PKC), we have isolated, cloned, and characterized a protein kinase from Colletotrichum trifolii, causal agent of alfalfa anthracnose. Though sequence analysis indicated conserved sequences in mammalian PKC genes, we were unable to induce activity of the fungal protein using known activators of PKC. Instead, we show that the C. trifolii gene, designated LIPK (lipidinduced protein kinase) is induced specifically by purified plant cutin or long-chain fatty acids which are monomeric constituents of cutin. PKC inhibitors prevented appressorium formation and, to a lesser extent, spore germination. Overexpression of LIPK resulted in multiple, abnormally shaped appressoria. Gene replacement of lipk yielded strains which were unable to develop appressoria and were unable to infect intact host plant tissue. However, these mutants were able to colonize host tissue following artificial wounding, resulting in typical anthracnose lesions. Taken together, these data indicate a central role in triggering infection structure formation for this protein kinase, which is induced specifically by components of the plant cuticle. Thus, the fungus is able to sense and use host surface chemistry to induce a protein kinase-mediated pathway that is required for pathogenic development.

Selected antiapoptotic genes were expressed in baker's yeast (Saccharomyces cerevisiae) to evaluate cytoprotective effects during oxidative stress. When exposed to treatments resulting in the generation of reactive oxygen species (ROS), including H(2)O(2), menadione, or heat shock, wild-type yeast died and exhibited apoptotic-like characteristics, consistent with previous studies. Yeast strains were generated expressing nematode ced-9, human bcl-2, or chicken bcl-xl genes. These transformants tolerated a range of oxidative stresses, did not display features associated with apoptosis, and remained viable under conditions that were lethal to wild-type yeast. Yeast strains expressing a mutant antiapoptotic gene (bcl-2 deltaalpha 5-6), known to be nonfunctional in mammalian cells, were unable to tolerate any of the ROS-generating insults. These data are the first report showing CED-9 has cytoprotective effects against oxidative stress, and add CED-9 to the list of Bcl-2 protein family members that modulate ROS-mediated programmed cell death. In addition, these data indicate that Bcl-2 family members protect wild-type yeast from physiological stresses. Taken together, these data support the concept of the broad evolutionary conservation and functional similarity of the apoptotic processes in eukaryotic organisms.

Colletotrichum trifolii is the causative organism of alfalfa anthracnose. We previously cloned and characterized the small prototypical G protein, Ras, of C. trifolii , which is involved in the signaling pathways that mediate interaction between the pathogen and its host. Transformants expressing constitutively active forms of Ras have growth medium-dependent phenotypes. In nutrient-rich media (e.g., yeast extract and peptone), the phenotype of the transformants was indistinguishable from that of the wild type. However, during nutrient starvation, the transformants lose polarity, have distended hyphae, and fail to sporulate and produce appressoria. Since peptone caused the phenotype to revert, amino acids were tested singly and in combination to identify the responsible amino acid(s). We found that 1.6 mM proline in the medium reverses the constitutively active Ras phenotype.

In the version of this Perspective originally published, the name of co-author Paul de Figueiredo was incorrect. This has now been corrected.