The crystal structure of necrosis- and ethylene-inducing protein 2 from the causal agent of cacao's Witches' Broom disease reveals key elements for its activity

The necrosis- and ethylene-inducing peptide 1 (NEP1)-like proteins (NLPs) are proteins secreted from bacteria, fungi and oomycetes, triggering immune responses and cell death in dicotyledonous plants. Genomic-scale studies of Moniliophthora perniciosa, the fungus that causes the Witches' Broom disease in cacao, which is a serious economic concern for South and Central American crops, have identified five members of this family (termed MpNEP1-5). Here, we show by RNA-seq that MpNEP2 is virtually the only NLP expressed during the fungus infection. The quantitative real-time polymerase chain reaction results revealed that MpNEP2 has an expression pattern that positively correlates with the necrotic symptoms, with MpNEP2 reaching its highest level of expression at the advanced necrotic stage. To improve our understanding of MpNEP2's molecular mechanism of action, we determined the crystallographic structure of MpNEP2 at 1.8 ? resolution, unveiling some key structural features. The implications of a cation coordination found in the crystal structure were explored, and we show that MpNEP2, in contrast to another previously described member of the NLP family, NLP(Pya) from Pythium aphanidermatum, does not depend on an ion to accomplish its necrosis- and electrolyte leakage-promoting activities. Results of site-directed mutagenesis experiments confirmed the importance of a negatively charged cavity and an unforeseen hydrophobic β-hairpin loop for MpNEP2 activity, thus offering a platform for compound design with implications for disease control. Electron paramagnetic resonance and fluorescence assays with MpNEP2 performed in the presence of lipid vesicles of different compositions showed no sign of interaction between the protein and the lipids, implying that MpNEP2 likely requires other anchoring elements from the membrane to promote cytolysis or send death signals.     

Oxalic acid is an elicitor of plant programmed cell death during Sclerotinia sclerotiorum disease development

Accumulating evidence supports the idea that necrotrophic plant pathogens interact with their hosts by controlling cell death. Sclerotinia sclerotiorum is a necrotrophic ascomycete fungus with a broad host range (>400 species). Previously, we established that oxalic acid (OA) is an important pathogenicity determinant of this fungus. In this report, we describe a mechanism by which oxalate contributes to the pathogenic success of this fungus; namely, that OA induces a programmed cell death (PCD) response in plant tissue that is required for disease development. This response exhibits features associated with mammalian apoptosis, including DNA laddering and TUNEL reactive cells. Fungal mutants deficient in OA production are nonpathogenic, and apoptotic-like characteristics are not observed following plant inoculation. The induction of PCD by OA is independent of the pH-reducing abilities of this organic acid, which is required for sclerotial development. Moreover, oxalate also induces increased reactive oxygen species (ROS) levels in the plant, which correlate to PCD. When ROS induction is inhibited, apoptotic-like cell death induced by OA does not occur. Taken together, we show that Sclerotinia spp.-secreted OA is an elicitor of PCD in plants and is responsible for induction of apoptotic-like features in the plant during disease development. This PCD is essential for fungal pathogenicity and involves ROS. Thus, OA appears to function by triggering in the plant pathways responsible for PCD. Further, OA secretion by Sclerotinia spp. is not directly toxic but, more subtly, may function as a signaling molecule.     

Tipping the balance: Sclerotinia sclerotiorum secreted oxalic acid suppresses host defenses by manipulating the host redox environment

Sclerotinia sclerotiorum is a necrotrophic ascomycete fungus with an extremely broad host range. This pathogen produces the non-specific phytotoxin and key pathogenicity factor, oxalic acid (OA). Our recent work indicated that this fungus and more specifically OA, can induce apoptotic-like programmed cell death (PCD) in plant hosts, this induction of PCD and disease requires generation of reactive oxygen species (ROS) in the host, a process triggered by fungal secreted OA. Conversely, during the initial stages of infection, OA also dampens the plant oxidative burst, an early host response generally associated with plant defense. This scenario presents a challenge regarding the mechanistic details of OA function; as OA both suppresses and induces host ROS during the compatible interaction. In the present study we generated transgenic plants expressing a redox-regulated GFP reporter. Results show that initially, Sclerotinia (via OA) generates a reducing environment in host cells that suppress host defense responses including the oxidative burst and callose deposition, akin to compatible biotrophic pathogens. Once infection is established however, this necrotroph induces the generation of plant ROS leading to PCD of host tissue, the result of which is of direct benefit to the pathogen. In contrast, a non-pathogenic OA-deficient mutant failed to alter host redox status. The mutant produced hypersensitive response-like features following host inoculation, including ROS induction, callose formation, restricted growth and cell death. These results indicate active recognition of the mutant and further point to suppression of defenses by the wild type necrotrophic fungus. Chemical reduction of host cells with dithiothreitol (DTT) or potassium oxalate (KOA) restored the ability of this mutant to cause disease. Thus, Sclerotinia uses a novel strategy involving regulation of host redox status to establish infection. These results address a long-standing issue involving the ability of OA to both inhibit and promote ROS to achieve pathogenic success.     

Transcriptomics and systems biology analysis in identification of specific pathways involved in cacao resistance and susceptibility to witches' broom disease

This study reports on expression analysis associated with molecular systems biology of cacao-Moniliophthora perniciosa interaction. Gene expression data were obtained for two cacao genotypes (TSH1188, resistant; Catongo, susceptible) challenged or not with the fungus M. perniciosa and collected at three time points through disease. Using expression analysis, we identified 154 and 227 genes that are differentially expressed in TSH1188 and Catongo, respectively. The expression of some of these genes was confirmed by RT-qPCR. Physical protein-protein interaction (PPPI) networks of Arabidopsis thaliana orthologous proteins corresponding to resistant and susceptible interactions were obtained followed by cluster and gene ontology analyses. The integrated analysis of gene expression and systems biology allowed designing a general scheme of major mechanisms associated with witches' broom disease resistance/susceptibility. In this sense, the TSH1188 cultivar shows strong production of ROS and elicitors at the beginning of the interaction with M. perniciosa followed by resistance signal propagation and ROS detoxification. On the other hand, the Catongo genotype displays defense mechanisms that include the synthesis of some defense molecules but without success in regards to elimination of the fungus. This phase is followed by the activation of protein metabolism which is achieved with the production of proteasome associated with autophagy as a precursor mechanism of PCD. This work also identifies candidate genes for further functional studies and for genetic mapping and marker assisted selection.     

Nep1‐like protein from Moniliophthora perniciosa induces a rapid proteome and metabolome reprogramming in cells of Nicotiana benthamiana

NEP1 (necrosis‐ and ethylene‐inducing peptide 1)‐like proteins (NLPs) have been identified in a variety of taxonomically unrelated plant pathogens and share a common characteristic of inducing responses of plant defense and cell death in dicotyledonous plants. Even though some aspects of NLP action have been well characterized, nothing is known about the global range of modifications in proteome and metabolome of NLP‐treated plant cells. Here, using both proteomic and metabolomic approaches we were able to identify the global molecular and biochemical changes in cells of Nicotiana benthamiana elicited by short‐term treatment with MpNEP2, a NLP of Moniliophthora perniciosa, the basidiomycete responsible for the witches' broom disease on cocoa (Theobroma cacao L.). Approximately 100 protein spots were collected from 2‐DE gels in each proteome, with one‐third showing more than twofold differences in the expression values. Fifty‐three such proteins were identified by mass spectrometry (MS)/MS and mapped into specific metabolic pathways and cellular processes. Most MpNEP2 upregulated proteins are involved in nucleotide‐binding function and oxidoreductase activity, whereas the downregulated proteins are mostly involved in glycolysis, response to stress and protein folding. Thirty metabolites were detected by gas spectrometry (GC)/MS and semi‐quantified, of which eleven showed significant differences between the treatments, including proline, alanine, myo‐inositol, ethylene, threonine and hydroxylamine. The global changes described affect the reduction‐oxidation reactions, ATP biosynthesis and key signaling molecules as calcium and hydrogen peroxide. These findings will help creating a broader understanding of NLP‐mediated cell death signaling in plants.     

Deficient plastidic fatty acid synthesis triggers cell death by modulating mitochondrial reactive oxygen species

Programmed cell death (PCD) is of fundamental importance to development and defense in animals and plants. In plants, a well-recognized form of PCD is hypersensitive response (HR) triggered by pathogens, which involves the generation of reactive oxygen species (ROS) and other signaling molecules. While the mitochondrion is a master regulator of PCD in animals, the chloroplast is known to regulate PCD in plants. Arabidopsis Mosaic Death 1 (MOD1), an enoyl-acyl carrier protein (ACP) reductase essential for fatty acid biosynthesis in chloroplasts, negatively regulates PCD in Arabidopsis. Here we report that PCD in mod1 results from accumulated ROS and can be suppressed by mutations in mitochondrial complex I components, and that the suppression is confirmed by pharmaceutical inhibition of the complex I-generated ROS. We further show that intact mitochondria are required for full HR and optimum disease resistance to the Pseudomonas syringae bacteria. These findings strongly indicate that the ROS generated in the electron transport chain in mitochondria plays a key role in triggering plant PCD and highlight an important role of the communication between chloroplast and mitochondrion in the control of PCD in plants.     

Comparative analysis of expressed genes from cacao meristems infected by Moniliophthora perniciosa

BACKGROUND AND AIMS: Witches' broom disease is caused by the hemibiotrophic basidiomycete Moniliophthora perniciosa, and is one of the most important diseases of cacao in the western hemisphere. Because very little is known about the global process of such disease development, expressed sequence tags (ESTs) were used to identify genes expressed during the Theobroma cacao-Moniliophthora perniciosa interaction. METHODS: Two cDNA libraries corresponding to the resistant (RT) and susceptible (SP) cacao-M. perniciosa interactions were constructed from total RNA, using the DB SMART Creator cDNA library kit (Clontech). Clones were randomly selected, sequenced from the 5' end and analysed using bioinformatics tools including in silico analysis of the differential gene expression. KEY RESULTS: A total of 6884 ESTs were generated from the RT and SP cDNA libraries. These ESTs were composed of 2585 singlets and 341 contigs for a total of 2926 non-redundant sequences. The redundancy of the libraries was low and their specificity high when compared with the few other cacao libraries already published. Sequence analysis allowed the assignment of a putative functional category for 54 % of sequences, whereas approx. 22 % of sequences corresponded to unknown function and approx. 24 % of sequences did not show any significant similarity with other proteins present in the database. Despite the similar overall distribution of the sequences in functional categories between the two libraries, qualitative differences were observed. Genes involved during the defence response to pathogen infection or in programmed cell death were identified, such as pathogenesis related-proteins, trypsin inhibitor or oxalate oxidase, and some of them showed an in silico differential expression between the resistant and the susceptible interactions. CONCLUSIONS: As far as is known this is the first EST resource from the cacao-M. perniciosa interaction and it is believed that it will provide a significant contribution to the understanding of the molecular mechanisms of the resistance and susceptibility of cacao to M. perniciosa, to develop strategies to control witches' broom, and as a source of polymorphism for molecular marker development and marker-assisted selection.     

Carbon source-dependent variation of acquired mutagen resistance of Moniliophthora perniciosa: similarities in natural and artificial systems

The basidiomycete Moniliophthora perniciosa causes Witches' Broom disease in Theobroma cacao. We studied the influence of carbon source on conditioning hyphae to oxidative stress agents (H(2)O(2), paraquat, 4NQO) and to UVC, toward the goal of assessing the ability of this pathogen to avoid plant defenses involving ROS. Cells exhibited increased resistance to H(2)O(2) when shifted from glucose to glycerol and from glycerol to glycerol. When exposed to paraquat, cells grown in fresh medium were always more resistant. Apparently glycerol and/or fresh media, but not old glucose media, up-regulate oxidative stress defenses in this fungus. For the mutagens UVC and 4NQO, whose prime action on DNA is not via ROS, change of carbon source did not elicit a clear change in sensitivity/resistance. These results correlate with expression of fungal genes that protect against ROS and with biochemical changes observed in infected cacao tissues, where glycerol and high amounts of ROS have been detected in green brooms.     

Programmed cell death suppression in transformed plant tissue by tomato cDNAs identified from an <Emphasis Type="Italic">Agrobacterium rhizogenes</Emphasis>-based functional screen

The genetic regulation of programmed cell death (PCD) is well characterized in animal systems, but largely unresolved in plants. This research was designed to identify plant genes that can suppress PCD triggered in plants by Fumonisin B1 (FB1). Agrobacterium rhizogenes was used to transform individual members of a cDNA library into tomato roots, which were then screened for resistance to FB1. Cellular changes elicited during FB1-induced PCD include chromatin condensation, fragmentation into pycnotic DNA bodies, TUNEL positive reactions, ROS accumulation, and eventual loss of membrane integrity. Several cDNA library members collectively overexpressed in a transformed root population revealed PCD suppressive action and were recovered by PCR. One of the FB1 suppressive genes was homologous to metallothionein, and shared sequence homology to the animal ortholog reported to suppress PCD through interference with formation or activity of reactive oxygen species (ROS). The metallothionein recovered in this screen suppressed ROS accumulation in FB1-treated roots and prevented symptoms of PCD. Anti-PCD genes recovered by this screen represent potential sources of resistance to PCD-dependent plant diseases, while the screen should be useful to identify genes capable of suppressing PCD triggered by other effectors, including those expressed by root pathogens during infection.     

DspA/E, a type III effector of Erwinia amylovora, is required for early rapid growth in Nicotiana benthamiana and causes NbSGT1-dependent cell death

DspA/E is a pathogenicity factor of Erwinia amylovora that is translocated into the plant cell cytoplasm through an Hrp type III secretion system. Transient expression of dspA/E in Nicotiana benthamiana or yeast induced cell death, as it does in N. tabacum and apple as described previously. DspA/E-induced cell death in N. benthamiana was not inhibited by coexpression of AvrPtoB of Pseudomonas syringae pv. tomato , which inhibits programmed cell death (PCD) induced by several other elicitors in plants. Silencing of NbSGT1 , the expression of which is required for PCD mediated by several resistance proteins of plants, prevented DspA/E-induced cell death in N. benthamiana. However, silencing of NbRAR1 , or two MAP kinase kinase genes, which are required for PCD associated with many resistance genes in plants, did not prevent cell death induced by DspA/E. Silencing of NbSGT1 also compromised non-host resistance against E. amylovora . E. amylovora grew rapidly within the first 24 h after infiltration in N. benthamiana , and DspA/E was required for this early rapid growth. However, bacterial cell numbers decreased after 24 h in TRV-vector-transformed plants, whereas a dspA/E mutant strain grew to high populations in NbSGT1 -silenced plants. Our results indicate that DspA/E enhances virulence of E. amylovora in N. benthamiana, but the bacteria are then recognized by the plant, resulting in PCD and death of bacterial cells or restriction of bacterial cell growth.     

Lesion mimic mutants: keys for deciphering cell death and defense pathways in plants?

The identification of several lesion mimic mutants (LMM) that misregulate cell death constitutes a powerful tool to unravel programmed cell death (PCD) pathways in plants, particularly the hypersensitive response (HR), a form of PCD associated with resistance to pathogens. Recently, the characterization of novel LMM has enabled genes that might regulate cell death programmes to be identified as well as the dissection of defense signaling pathways and of crosstalk between multiple pathways in ways that might not be possible by studying the responses of wild-type plants to pathogens.     

Ornithine-delta-aminotransferase and proline dehydrogenase genes play a role in non-host disease resistance by regulating pyrroline-5-carboxylate metabolism-induced hypersensitive response

Non-host disease resistance involves the production of hypersensitive response (HR), a programmed cell death (PCD) that occurs at the site of pathogen infection. Plant mitochondrial reactive oxygen species (ROS) production and red-ox changes play a major role in regulating such cell death. Proline catabolism reactions, especially pyrroline-5-carboxylate (P5C) accumulation, are known to produce ROS and contribute to cell death. Here we studied important genes related to proline synthesis and catabolism in the defence against host and non-host strains of Pseudomonas syringae in Nicotiana benthamiana and Arabidopsis. Our results show that ornithine delta-aminotransferase (δOAT) and proline dehydrogenases (ProDH1 and ProDH2) are involved in the defence against non-host pathogens. Silencing of these genes in N. benthamiana delayed occurrence of HR and favoured non-host pathogen growth. Arabidopsis mutants for these genes compromised non-host resistance and showed a decrease in non-host pathogen-induced ROS. Some of the genes involved in proline metabolism were also induced by a pathogen-carrying avirulence gene, indicating that proline metabolism is influenced during effector-triggered immunity (ETI). Our results demonstrate that δOAT and ProDH enzyme-mediated steps produce ROS in mitochondria and regulate non-host HR, thus contributing to non-host resistance in plants.     

StCDPK5 confers resistance to late blight pathogen but increases susceptibility to early blight pathogen in potato via reactive oxygen species burst

? Potato (Solanum tuberosum) calcium-dependent protein kinase (StCDPK5) has been shown to phosphorylate the N-terminal region of plasma membrane RBOH (respiratory burst oxidase homolog) proteins, and participate in StRBOHB-mediated reactive oxygen species (ROS) burst. The constitutively active form, StCDPK5VK, provides a useful tool for gain-of-function analysis of RBOH in defense responses. ? StCDPK5- and StCDPK5VK-green fluorescent protein fusion proteins were predominantly targeted to the plasma membrane, and conditional expression of StCDPK5VK activated StRBOHA-D. The interaction was confirmed by bimolecular fluorescence complementation assay. We generated transgenic potato plants containing StCDPK5VK under the control of a pathogen-inducible promoter to investigate the role of ROS burst on defense responses to blight pathogens. ? Virulent isolates of the late blight pathogen Phytophthora infestans and the early blight pathogen Alternaria solani induced hypersensitive response-like cell death accompanied by ROS production at the infection sites of transgenic plants. Transgenic plants showed resistance to the near-obligate hemibiotrophic pathogen P.?infestans and, by contrast, increased susceptibility to the necrotrophic pathogen A.?solani. ? These results indicate that RBOH-dependent ROS contribute to basal defense against near-obligate pathogens, but have a negative role in resistance or have a positive role in expansion of disease lesions caused by necrotrophic pathogens.     

The effect of TAO expression on PCD-like phenomenon development and drug resistance in Trypanosoma brucei

Drug resistance in Trypanosoma brucei causes severe problems for people and domestic animals, but molecular mechanisms of the resistance are not well known. Programmed cell death (PCD) is a fundamental process in both multicellular and unicellular organisms, and it is speculated to be one of the important factors contributing to the emergence of drug resistance. We have previously reported that the expression of TAO appears to play a role in the inhibition of the PCD-like phenomenon development in T. brucei. In this study, to ascertain the correlation between the development of the PCD-like phenomenon and the expression of TAO in T. brucei, we genetically engineered T. brucei for conditional over-expression of the TAO gene. TAO over-expressing transgenic T. brucei was refractory to the development of the PCD-like phenomenon compared to the wild-type, indicating that expression of TAO might have a regulatory role on PCD development. Furthermore, the transgenic cells showed resistance to suramin and antrycide. We postulated that intracellular reactive oxygen species (ROS) may be involved in the mechanism of resistance to antrycide because augmentation of ROS in transgenic cells was lower than that in the wild-type cells following treatment with antrycide. These results suggest a possible correlation of PCD to drug resistance in T. brucei.     

A local accumulation of the Ralstonia solanacearum PopA protein in transgenic tobacco renders a compatible plant–pathogen interaction incompatible

Plants activate disease resistance responses when they recognize pathogen-derived molecules (elicitors). Frequently, recognition results in a hypersensitive response (HR), which is characterized by local host cell death at the infection site. Here we describe a genetic engineering approach to generate an HR in plants, whether or not an invading micro-organism produces a recognized elicitor. To that aim we created transgenic tobacco plants in which the pathogen-inducible promoter of the hsr203J gene from tobacco controls the expression of the popA elicitor gene from Ralstonia solanacearum. Because PopA itself also induces the hsr203J promoter, transgenic plants rapidly accumulate the bacterial elicitor in the pathogen infection sites. The elicitor becomes converted in plant tissues into its fully active derivatives PopA1-PopA3, showing that the previously observed processing events are not dependent on the bacterial type III secretion system. The outcome of induced PopA accumulation is a localized HR and a high degree of resistance of the transgenic plants to an oomycete pathogen. The system is functional in hybrids between different tobacco varieties, and we show that the engineered resistance, but not the associated cell death, is dependent on the salicylic acid signalling cascade. Although the approach is powerful in generating oomycete resistance, the induced HR might affect plant health. Its application thus requires a careful selection of individual transgenic lines and trials with various pathogens.     

Endophytic and pathogenic isolates of the cacao fungal pathogen Moniliophthora perniciosa (Tricholomataceae) are indistinguishable based on genetic and physiological analysis

We evaluated the genetic and physiological variability of Moniliophthora perniciosa obtained from healthy and diseased branches of cacao (Theobroma cacao) plants. The diversity of the isolates was evaluated by RAPD technique and by studies of virulence and exoenzyme production. The genetic variability of endophytic and pathogenic M. perniciosa was evaluated in association with pathogenicity assays. RAPD analysis showed eight genetic groups, which were not related to plant disease status (healthy versus diseased branches). Isolates from cacao were included in three groups, excluding isolates from other host plants. Pathogenicity and enzyme analysis showed that the virulence of the isolates is not related to exoenzyme production. This is the first evidence that M. perniciosa colonizes healthy parenchymatic tissues, showing that endophytic behavior may occur in this species.     

Chloroplasts regulate leaf senescence: delayed senescence in transgenic ndhF-defective tobacco

Mitochondrial involvement has not been identified in the programmed cell death (PCD) of leaf senescence which suggests that processes such as those involving reactive oxygen species (ROS) are controlled by chloroplasts. We report that transgenic tobacco (DeltandhF), with the plastid ndhF gene knocked-out, shows low levels of the plastid Ndh complex, homologous to mitochondrial complex I, and more than a 30-day-delay in leaf senescence with respect to wt. The comparison of activities and protein levels and analyses of genetic and phenotypic traits of wtxDeltandhF crosses indicate that regulatory roles of mitochondria in animal PCD are assumed by chloroplasts in leaf senescence. The Ndh complex would increase the reduction level of electron transporters and the generation of ROS. Chloroplastic control of leaf senescence provides a nonclassical model of PCD and reveals an unexpected role of the plastid ndh genes that are present in most higher plants.     

Characterization of <Emphasis Type="Italic">Rubus fruticosus</Emphasis> mitochondria and salicylic acid inhibition of reactive oxygen species generation at Complex III/Q cycle: potential implications for hypersensitive response in plants

In addition to adenosine triphosphate (ATP) production, mitochondria have been implicated in the regulation of several physiological responses in plants, such as programmed cell death (PCD) activation. Salicylic acid (SA) and reactive oxygen species (ROS) are essential signaling molecules involved in such physiological responses; however, the mechanisms by which they act remain unknown. In non-photosynthesizing tissues, mitochondria appear to serve as the main source of ROS generation. Evidence suggests that SA and ROS could regulate plant PCD through a synergistic mechanism that involves mitochondria. Herein, we isolate and characterize the mitochondria from non-photosynthesizing cell suspension cultures of Rubus fruticosus. Furthermore, we assess the primary site of ROS generation and the effects of SA on isolated organelles. Mitochondrial Complex III was found to be the major source of ROS generation in this model. In addition, we discovered that SA inhibits the electron transport chain by inactivating the semiquinone radical during the Q cycle. Computational analyses confirmed the experimental data, and a mechanism for this action is proposed.     

Redox regulation in plant programmed cell death

Programmed cell death (PCD) is a genetically controlled process described both in eukaryotic and prokaryotic organisms. Even if it is clear that PCD occurs in plants, in response to various developmental and environmental stimuli, the signalling pathways involved in the triggering of this cell suicide remain to be characterized. In this review, the main similarities and differences in the players involved in plant and animal PCD are outlined. Particular attention is paid to the role of reactive oxygen species (ROS) as key inducers of PCD in plants. The involvement of different kinds of ROS, different sites of ROS production, as well as their interaction with other molecules, is crucial in activating PCD in response to specific stimuli. Moreover, the importance is stressed on the balance between ROS production and scavenging, in various cell compartments, for the activation of specific steps in the signalling pathways triggering this cell suicide process. The review focuses on the complexity of the interplay between ROS and antioxidant molecules and enzymes in determining the most suitable redox environment required for the occurrence of different forms of PCD.