Time for Chocolate: Current Understanding and New Perspectives on Cacao Witches’ Broom Disease Research

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The mitochondrial alternative oxidase of Moniliophthora perniciosa and Moniliophthora roreri : its possible function in fungal development and pathogenesis

Orientadores: Gonçalo Amarante Guimarães Pereira, Johana Rincones PerezTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: Moniliophihora perniciosa e Monüiophthora roreri são basidiomicetos fitopatogênicos. causadores de duas das mais devastadoras doenças fúngicas do cacaueiro: a Vassoura de bruxa e a Frosty Pod Rot (Momlíase), respectivamente. Apesar do grande impacto socioeconómico dessas doenças, aspectos importantes relacionados ao metabolismo destes patógenos ainda não são suficientemente compreendidos O estudo do metabolismo mitocondrial é particularmente relevante, pois compreende processos celulares vitais para a sobrevivência do organismo, como a regulação da produção de energia (ATP) e a manutenção do balanço redox da célula. Este trabalho teve como objetivo entender a importância da enzima mitocondrial oxidase alternativa (AOX) no metabolismo e desenvolvimento dos fungos M. perniciosa e M roreri- A AOX é uma oxidase terminal capaz de realizar o transporte de elétrons mitocondrial, catalisando a redução do oxigênio a água, de maneira desacoplada da síntese de ATP. Em M. perniciosa, a expressão do gene aox (Mp-aox) mostrou-se bastante relacionada ao ciclo de vida hemibiotrófico deste fungo. Níveis de expressão relativamente elevados deste gene foram observados no micélio biotrófico cultivado in vitro. De acordo, grande sensibilidade à inibição da via alternativa também foi verificada nesta fase micelial, sugerindo que a AOX tem participação no desenvolvimento deste primeiro estágio de M. perniciosa. Curiosamente, a inibição in vitro da cadeia respiratória principal retardou a transição da fase biotrófica para a necrotrófíca. Além disso, o uso combinado de inibidores da via principal e da AOX resultou em total inibição do desenvolvimento do fungo in vitro como também preveniu o estabelecimento da doença em plântulas de cacau. Com base nesses dados, o presente trabalho sugere um modelo no qual a participação diferencial das vias de transporte de elétrons (principal e alternativa) regula o desenvolvimento do micélio biotrófico e a transição de fase de M. perniciosa. Este é o primeiro trabalho em que se apresentam fortes evidências para uma função da AOX no desenvolvimento in planta e ciclo de vida de um fungo fítopatogênico. Como em M. perniciosa, o gene aox de M. roreri (Mr-aox) foi induzido em resposta ao fungicida azoxistrobina (inibidor do complexo III), sugerindo que a AOX tem participação na proteção do patògeno contra o estresse oxidativo gerado pela inibição da via principal- Apesar da indução do gene Mr-aox, M. roreri mostrou-se consideravelmente sensível ao fungicida. Deste modo, é possível que a ativação da AOX em M. roreri não seja suficiente para contornar os efeitos tóxicos da azoxistrobina e, consequentemente, que este fungo seja mais suscetível ao estresse oxidativo gerado pelo inibidor. Finalmente, a adição conjunta da azoxistrobina com um inibidor da AOX inibiu completamente o desenvolvimento in vitro de M. roreri. Com base nos resultados apresentados, sugere-se que a AOX tenha uma função relevante no metabolismo destes patógenos e que a inibição conjunta das vias principal e alternativa seja uma estratégia em potencial para o controle das doenças Vassoura de bruxa e Frosty PodRot.Abstract: Moniliophthora perniciosa and Moniliophthora roreri are phytopathogenic basidiomycetes, which cause two of the most devastating fungal diseases of cacao: the Witches' Broom and Frosty Pod Rot diseases, respectively. Despite the great socioeconomic impact of the diseases, important aspects of the metabolism of these pathogens are not sufficiently understood. The mitochondrial metabolism is particularly important since it is associated with many cellular processes of crucial importance for the organism survival, such as the regulation of energy production (ATP) and the maintenance of the cellular redox balance. This study aimed to understand the importance of the mitochondrial enzyme alternative oxidase (AOX) in the metabolism and development of the cacao pathogens M perniciosa and M. roreri. AOX is a non-phosporylating ubiquinol oxidase which catalyzes the reduction of molecular oxygen to water. In M. perniciosa, the expression of aox gene {Mp-aox) was closely related to the hemibiotrophic lifestyle of this fungus. High levels of Mp-aox transcripts were observed in the biotrophic mycelium and, accordingly, it showed an elevated sensitivity to AOX inhibitors, suggesting that AOX has a role in the development of the biotrophic phase. Interestingly, the in vitro inhibition of the cytochrome-dependent pathway prevented the transition from biotrophy to necrotrophy. Furthermore, the combined use of an inhibitor of the cytochrome pathway with an AOX inhibitor completely impaired the in vitro fungal growth as well as prevented the establishment of the disease in cacao seedlings. Based on these data, this study suggests a model in which the involvement of the different pathways of electron transfer (cytochrome and alternative routes) regulates the development of the biotrophic mycelium and the phase transition of M. perniciosa in planta. This is the first report presenting strong evidences for a role of AOX during the in pfanta development and life cycle of a phytopathogenic fijngus. Like M. perniciosa, the aox gene of M. roreri {Mr-aox) is up-regulated after exposure of the fungal mycelium to the fungicide azoxystrobin (a potent inhibitor of complex III}, suggesting that AOX plays a protective role against the deleterious oxidative stress produced by the compound. However, even with the increased expression of Mr-aox, M. roreri growth was highly sensitive to the fungicide. Therefore, it is possible that AOX activation was not enough to overcome the toxic effects of the cytochrome pathway inhibition and, consequently, M. roreri presented a high susceptibility to oxidative stress. Finally, the combined application of azoxystrobin and an AOX inhibitor completely impaired the in vitro development of M. roreri Thus., based on these results, we suggest that AOX has a role in the metabolism of these cacao pathogens and the concomitant inhibition of both cytochrome and alternative pathways may be an efficient strategy for the control of Witches' Broom and Frosty Pod Rot diseases.DoutoradoGenetica de MicroorganismosDoutor em Genetica e Biologia Molecula

Time for chocolate: current understanding and new perspectives on cacao witches’ broom disease research

Theobroma cacao is a tropical understory tree that is one of the most important perennial crops in agriculture. Treasured by ancient civilizations in Mesoamerica for over 3,000 years, the cocoa bean now supports a multibillion-dollar industry that is involved in the production and commercialization of chocolate, a treat appreciated worldwide. The cacao tree is originally from the Amazon rainforest and is currently grown in more than 50 countries throughout the humid tropics, serving as a major source of income for over 40 million people. Each year, more than 3 million tons of cocoa beans are produced, mostly by smallholder farmers in areas of high biodiversity. Notably, the cacao tree does not require direct sunlight and naturally grows under the canopy of other, taller trees. This characteristic often encourages farmers to preserve existing forests and to plant additional trees to shelter their cacao plants [1], thereby reducing the environmental impacts of cacao cultivation. Despite its great importance, the cacao tree is affected by a number of untreatable diseases that reduce fruit production and threaten our global supply of cacao. Among them, witches' broom disease (WBD) stands out as one of the most severe problems that affect this crop, accounting for production losses of up to 90%1110CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP475535/2013-809/50119-

The <i>Moniliophthora perniciosa</i> life cycle in <i>Theobroma cacao</i>.

<p>Infection begins when fungal basidiospores penetrate the plant through stomata or wounds. In the first stage of the disease, <i>M</i>. <i>perniciosa</i> develops as a swollen monokaryotic mycelium that grows exclusively in the extracellular space of the living plant tissue. Infection of shoots induces drastic morphological alterations resulting in the characteristic “green broom” structure, though infection can also occur in other tissues (fruits and flowers). After one to three months of biotrophic infection, necrosis of the plant tissue occurs, giving rise to the “dry broom” structure. Necrotic tissue is colonized intracellularly by thin dikaryotic mycelium, which is characterized by the presence of clamp connections—a cross structure formed by hyphal cells that ensures the presence of two nuclei in each fungal cell. After alternating rainy and dry periods, basidiomata are formed from necrotrophic hyphae, completing the pathogen life cycle. Illustrations by Diana Carneiro.</p

The pathogenic lifestyle of <i>M</i>. <i>perniciosa</i> is an exception within the Marasmiaceae family of basidiomycetes, which is mostly composed of saprotrophic litter and wood-decomposing fungi.

<p>The genus <i>Moniliophthora</i> includes the hemibiotrophic sister species <i>M</i>. <i>perniciosa</i> and <i>M</i>. <i>roreri</i>, the two major pathogens of <i>Theobroma cacao</i>. Notably, it also encompasses a still poorly characterized grass endophyte, suggesting that the pathogenic lifestyle of <i>M</i>. <i>perniciosa</i> may have evolved from an endophyte ancestral. The tree was constructed based on Bayesian inference using regions of the genes 25S, 18S, ITS/5.8S and Rbp1 (large fragment of the RNA polymerase II). Sequences were retrieved from Aime & Phillips-Mora (2005) [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005130#ppat.1005130.ref005" target="_blank">5</a>] and Matheny et al. (2006) [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005130#ppat.1005130.ref034" target="_blank">34</a>]. Numbers next to the branches represent the posterior probabilities. The species <i>Schizophyllum radiatum</i> was used as outgroup.</p

Suppression of Plant Immunity by Fungal Chitinase-like Effectors

Crop diseases caused by fungi constitute one of the most important problems in agriculture, posing a serious threat to food security [1]. To establish infection, phytopathogens interfere with plant immune responses [2, 3]. However, strategies to promote virulence employed by fungal pathogens, especially non-model organisms, remain elusive [4], mainly because fungi are more complex and difficult to study when compared to the better-characterized bacterial pathogens. Equally incomplete is our understanding of the birth of microbial virulence effectors. Here, we show that the cacao pathogen Moniliophthora perniciosa evolved an enzymatically inactive chitinase (MpChi) that functions as a putative pathogenicity factor. MpChi is among the most highly expressed fungal genes during the biotrophic interaction with cacao and encodes a chitinase with mutations that abolish its enzymatic activity. Despite the lack of chitinolytic activity, MpChi retains substrate binding specificity and prevents chitin-triggered immunity by sequestering immunogenic chitin fragments. Remarkably, its sister species M. roreri encodes a second non-orthologous catalytically impaired chitinase with equivalent function. Thus, a class of conserved enzymes independently evolved as putative virulence factors in these fungi. In addition to unveiling a strategy of host immune suppression by fungal pathogens, our results demonstrate that the neofunctionalization of enzymes may be an evolutionary pathway for the rise of new virulence factors in fungi. We anticipate that analogous strategies are likely employed by other pathogens. Fiorin et al. demonstrate that two fungal pathogens of cacao independently evolved catalytically dead chitinases that bind to chitin and prevent elicitation of plant immunity. The study exemplifies how pathogens may evolve effectors by repurposing the functions of enzymes that are conserved throughout evolution.</p

Suppression of Plant Immunity by Fungal Chitinase-like Effectors

Crop diseases caused by fungi constitute one of the most important problems in agriculture, posing a serious threat to food security [1]. To establish infection, phytopathogens interfere with plant immune responses [2, 3]. However, strategies to promote virulence employed by fungal pathogens, especially non-model organisms, remain elusive [4], mainly because fungi are more complex and difficult to study when compared to the better-characterized bacterial pathogens. Equally incomplete is our understanding of the birth of microbial virulence effectors. Here, we show that the cacao pathogen Moniliophthora perniciosa evolved an enzymatically inactive chitinase (MpChi) that functions as a putative pathogenicity factor. MpChi is among the most highly expressed fungal genes during the biotrophic interaction with cacao and encodes a chitinase with mutations that abolish its enzymatic activity. Despite the lack of chitinolytic activity, MpChi retains substrate binding specificity and prevents chitin-triggered immunity by sequestering immunogenic chitin fragments. Remarkably, its sister species M. roreri encodes a second non-orthologous catalytically impaired chitinase with equivalent function. Thus, a class of conserved enzymes independently evolved as putative virulence factors in these fungi. In addition to unveiling a strategy of host immune suppression by fungal pathogens, our results demonstrate that the neofunctionalization of enzymes may be an evolutionary pathway for the rise of new virulence factors in fungi. We anticipate that analogous strategies are likely employed by other pathogens. Fiorin et al. demonstrate that two fungal pathogens of cacao independently evolved catalytically dead chitinases that bind to chitin and prevent elicitation of plant immunity. The study exemplifies how pathogens may evolve effectors by repurposing the functions of enzymes that are conserved throughout evolution.</p

Loss of function of a DMR6 ortholog in tomato confers broad-spectrum disease resistance

Plant diseases are among the major causes of crop yield losses around the world. To confer disease resistance, conventional breeding relies on the deployment of single resistance (R) genes. However, this strategy has been easily overcome by constantly evolving pathogens. Disabling susceptibility (S) genes is a promising alternative to R genes in breeding programs, as it usually offers durable and broad-spectrum disease resistance. In Arabidopsis, the S gene DMR6 (AtDMR6) encodes an enzyme identified as a susceptibility factor to bacterial and oomycete pathogens. Here, we present a model-to-crop translational work in which we characterize two AtDMR6 orthologs in tomato, SlDMR6-1 and SlDMR6-2. We show that SlDMR6-1, but not SlDMR6-2, is up-regulated by pathogen infection. In agreement, Sldmr6-1 mutants display enhanced resistance against different classes of pathogens, such as bacteria, oomycete, and fungi. Notably, disease resistance correlates with increased salicylic acid (SA) levels and transcriptional activation of immune responses. Furthermore, we demonstrate that SlDMR6-1 and SlDMR6-2 display SA-5 hydroxylase activity, thus contributing to the elucidation of the enzymatic function of DMR6. We then propose that SlDMR6 duplication in tomato resulted in subsequent subfunctionalization, in which SlDMR6-2 specialized in balancing SA levels in flowers/fruits, while SlDMR6-1 conserved the ability to fine-tune SA levels during pathogen infection of the plant vegetative tissues. Overall, this work not only corroborates a mechanism underlying SA homeostasis in plants, but also presents a promising strategy for engineering broad-spectrum and durable disease resistance in crops

High-resolution Transcript Profiling Of The Atypical Biotrophic Interaction Between Theobroma Cacao And The Fungal Pathogen Moniliophthora Perniciosa.

Witches' broom disease (WBD), caused by the hemibiotrophic fungus Moniliophthora perniciosa, is one of the most devastating diseases of Theobroma cacao, the chocolate tree. In contrast to other hemibiotrophic interactions, the WBD biotrophic stage lasts for months and is responsible for the most distinctive symptoms of the disease, which comprise drastic morphological changes in the infected shoots. Here, we used the dual RNA-seq approach to simultaneously assess the transcriptomes of cacao and M. perniciosa during their peculiar biotrophic interaction. Infection with M. perniciosa triggers massive metabolic reprogramming in the diseased tissues. Although apparently vigorous, the infected shoots are energetically expensive structures characterized by the induction of ineffective defense responses and by a clear carbon deprivation signature. Remarkably, the infection culminates in the establishment of a senescence process in the host, which signals the end of the WBD biotrophic stage. We analyzed the pathogen's transcriptome in unprecedented detail and thereby characterized the fungal nutritional and infection strategies during WBD and identified putative virulence effectors. Interestingly, M. perniciosa biotrophic mycelia develop as long-term parasites that orchestrate changes in plant metabolism to increase the availability of soluble nutrients before plant death. Collectively, our results provide unique insight into an intriguing tropical disease and advance our understanding of the development of (hemi)biotrophic plant-pathogen interactions.264245-6