Identification and Characterization of In planta–Expressed Secreted Effector Proteins from Magnaporthe oryzae That Induce Cell Death in Rice

Interactions between rice and Magnaporthe oryzae involve the recognition of cellular components and the exchange of complex molecular signals from both partners. How these interactions occur in rice cells is still elusive. We employed robust-long serial analysis of gene expression, massively parallel signature sequencing, and sequencing by synthesis to examine transcriptome profiles of infected rice leaves. A total of 6,413 in planta–expressed fungal genes, including 851 genes encoding predicted effector proteins, were identified. We used a protoplast transient expression system to assess 42 of the predicted effector proteins for the ability to induce plant cell death. Ectopic expression assays identified five novel effectors that induced host cell death only when they contained the signal peptide for secretion to the extracellular space. Four of them induced cell death in Nicotiana benthamiana. Although the five effectors are highly diverse in their sequences, the physiological basis of cell death induced by each was similar. This study demonstrates that our integrative genomic approach is effective for the identification of in planta–expressed cell death–inducing effectors from M. oryzae that may play an important role facilitating colonization and fungal growth during infection.

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Secretome Analysis and In Planta Expression of Salivary Proteins Identify Candidate Effectors from the Brown Planthopper Nilaparvata lugens

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-05-18-0122-r ◽

2019 ◽

Vol 32 (2) ◽

pp. 227-239 ◽

Cited By ~ 10

Author(s):

Weiwei Rao ◽

Xiaohong Zheng ◽

Bingfang Liu ◽

Qin Guo ◽

Jianping Guo ◽

...

Keyword(s):

Cell Death ◽

Expression Analysis ◽

Plant Pathogens ◽

Critical Role ◽

Brown Planthopper ◽

Ectopic Expression ◽

Nilaparvata Lugens ◽

Effector Proteins ◽

Salivary Proteins ◽

Secretome Analysis

The brown planthopper (BPH), Nilaparvata lugens (Stål), is a phloem sap-feeding insect. During feeding on rice plants, BPH secretes salivary proteins with potential effector functions, which may play a critical role in the plant–insect interactions. However, a limited number of BPH effector proteins have been identified to date. Here, we sequenced the salivary gland transcriptomes of five BPH populations and subsequently established a N. lugens secretome consisting of 1,140 protein-encoding genes. Secretome analysis revealed the presence of both conserved and rapidly evolving salivary proteins. A screen for potential effectors that elicit responses in the plant was performed via the transient expression analysis of 64 BPH salivary proteins in Nicotiana benthamiana leaves and rice protoplasts. The salivary proteins Nl12, Nl16, Nl28, and Nl43 induced cell death, whereas Nl40 induced chlorosis and Nl32 induced a dwarf phenotype in N. benthamiana, indicating effector properties of these proteins. Ectopic expression of the six salivary proteins in N. benthamiana upregulated expression of defense-related genes and callose deposition. Tissue expression analysis showed a higher expression level of the six candidate effectors in salivary glands than in other tissues. Subcellular localization and analysis of the domain required for cell death showed a diverse structure of the six effectors. Nl28, Nl40, and Nl43 are N. lugens specific; in contrast, Nl12, Nl16, and Nl32 are conserved among insects. The Nl40 family has numerous isoforms produced by alternative splicing, exemplifying rapid evolution and expansion of effector proteins in the BPH. Our results suggest a potential large effector repertoire in BPH and a higher level of effector conservation exist in BPH compared with that in plant pathogens.

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Comprehensive analysis of Verticillium nonalfalfae in silico secretome uncovers putative effector proteins expressed during hop invasion

10.1101/237255 ◽

2017 ◽

Author(s):

Kristina Marton ◽

Marko Flajšman ◽

Sebastjan Radišek ◽

Katarina Košmelj ◽

Jernej Jakše ◽

...

Keyword(s):

Verticillium Wilt ◽

Sequence Similarity ◽

Vascular Plant ◽

Xylem Sap ◽

Fungal Growth ◽

Effector Proteins ◽

Specific Gene ◽

In Planta ◽

Verticillium Nonalfalfae ◽

Spatio Temporal

AbstractBackgroundThe vascular plant pathogen Verticillium nonalfalfae causes Verticillium wilt in several important crops. VnaSSP4.2 was recently discovered as a V. nonalfalfae virulence effector protein in the xylem sap of infected hop. Here, we expanded our search for candidate secreted effector proteins (CSEPs) in the V. nonalfalfae predicted secretome using a bioinformatic pipeline built on V. nonalfalfae genome data, RNA-Seq and proteomic studies of the interaction with hop.ResultsThe secretome, rich in carbohydrate active enzymes, proteases, redox proteins and proteins involved in secondary metabolism, cellular processing and signaling, includes 263 CSEPs. Several homologs of known fungal effectors (LysM, NLPs, Hce2, Cerato-platanins, Cyanovirin-N lectins, hydrophobins and CFEM domain containing proteins) and avirulence determinants in the PHI database (Avr-Pita1 and MgSM1) were found. The majority of CSEPs were non-annotated and were narrowed down to 44 top priority candidates based on their likelihood of being effectors. These were examined by spatio-temporal gene expression profiling of infected hop. Among the highest in planta expressed CSEPs, five deletion mutants were tested in pathogenicity assays. A deletion mutant of VnaUn.279, a lethal pathotype specific gene with sequence similarity to SAM-dependent methyltransferase (LaeA), had lower infectivity and showed highly reduced virulence, but no changes in morphology, fungal growth or conidiation were observed.ConclusionsSeveral putative secreted effector proteins that probably contribute to V. nonalfalfae colonization of hop were identified in this study. Among them, LaeA gene homolog was found to act as a potential novel virulence effector of V. nonalfalfae. The combined results will serve for future characterization of V. nonalfalfae effectors, which will advance our understanding of Verticillium wilt disease.

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Transcriptome Profiling of the Rice Blast Fungus Magnaporthe oryzae and Its Host Oryza sativa During Infection

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-07-19-0207-a ◽

2020 ◽

Vol 33 (2) ◽

pp. 141-144 ◽

Cited By ~ 2

Author(s):

Jongbum Jeon ◽

Gir-Won Lee ◽

Ki-Tae Kim ◽

Sook-Young Park ◽

Seongbeom Kim ◽

...

Keyword(s):

Oryza Sativa ◽

Magnaporthe Oryzae ◽

Fungal Pathogen ◽

Rice Blast ◽

Transcriptome Profiling ◽

Transcriptome Data ◽

Highly Qualified ◽

In Planta ◽

Microbe Interactions ◽

Fungal Genes

The rice blast (fungal pathogen: Magnaporthe oryzae and host: Oryza sativa) is one of the most important model pathosystems for understanding plant–microbe interactions. Although both genome sequences were published as the first cases of pathogen and host, only a few in planta transcriptome data during infection are available. Due to technical difficulties, previously reported fungal transcriptome data are not highly qualified to comprehensively profile the expression of fungal genes during infection. Here, we report the high-quality transcriptomes of M. oryzae and rice during infection using a sheath infection-based RNA sequencing approach. This comprehensive expression profiling of the fungal pathogen and its host will provide a better platform for understanding the plant–microbe interactions at the genomic level and serve as a valuable resource for the research community.

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Functional Identification of Novel Cell Death-inducing Effector Proteins from Magnaporthe oryzae

Rice ◽

10.1186/s12284-019-0312-z ◽

2019 ◽

Vol 12 (1) ◽

Cited By ~ 3

Author(s):

Xinrui Guo ◽

Debin Zhong ◽

Wei Xie ◽

Yanhua He ◽

Yueqin Zheng ◽

...

Keyword(s):

Cell Death ◽

Magnaporthe Oryzae ◽

Effector Proteins ◽

Functional Identification

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Protective role of p21(Waf1/Cip1) against prostaglandin A2-mediated apoptosis of human colorectal carcinoma cells.

Molecular and Cellular Biology ◽

10.1128/mcb.16.12.6654 ◽

1996 ◽

Vol 16 (12) ◽

pp. 6654-6660 ◽

Cited By ~ 120

Author(s):

M Gorospe ◽

X Wang ◽

K Z Guyton ◽

N J Holbrook

Keyword(s):

Cell Death ◽

Colorectal Carcinoma ◽

Expression System ◽

Ectopic Expression ◽

Growth Arrest ◽

Protective Role ◽

Cellular Growth ◽

Human Colorectal Carcinoma ◽

P21 Waf1 ◽

Prostaglandin A2

Prostaglandin A2 (PGA2) suppresses tumor growth in vivo, is potently antiproliferative in vitro, and is a model drug for the study of the mammalian stress response. Our previous studies using breast carcinoma MCF-7 cells suggested that p21(Waf1/Cip1) induction enabled cells to survive PGA2 exposure. Indeed, the marked sensitivity of human colorectal carcinoma RKO cells to the cytotoxicity of PGA2 is known to be associated with a lack of a PGA2-mediated increase in p21(Waf1/Cip1) expression, inhibition of cyclin-dependent kinase activity, and growth arrest. To determine if cell death following exposure to PGA2 could be prevented by forcing the expression of p21(Waf1/Cip1) in RKO cells, we utilized an adenoviral vector-based expression system. We demonstrate that ectopic expression of p21(Waf1/Cip1) largely rescued RKO cells from PGA2-induced apoptotic cell death, directly implicating p21(Waf1/Cip1) as a determinant of the cellular outcome (survival versus death) following exposure to PGA2. To discern whether p21(Waf1/Cip1)-mediated protection operates through the implementation of cellular growth arrest, other growth-inhibitory treatments were studied for the ability to attenuate PGA2-induced cell death. Neither serum depletion nor suramin (a growth factor receptor antagonist) protected RKO cells against PGA2 cytotoxicity, and neither induced p21(Waf1/Cip1) expression. Mimosine, however, enhanced p21(Waf1/Cip1) expression, completely inhibited RKO cell proliferation, and exerted marked protection against a subsequent PGA2 challenge. Taken together, our results directly demonstrate a protective role for p21(Waf1/Cip1) during PGA2 cellular stress and provide strong evidence that the implementation of cellular growth arrest contributes to this protective influence.

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Expression of putative effectors of different Xylella fastidiosa subspecies/strains reveals recognition and defense activation in various model plants

10.1101/2021.05.27.445625 ◽

2021 ◽

Author(s):

Matthaios Sertedakis ◽

Konstantinos Kotsaridis ◽

Dimitra Tsakiri ◽

Ana Dominguez-Ferreras ◽

Vardis Ntoukakis ◽

...

Keyword(s):

Cell Death ◽

Host Defense ◽

Serine Proteases ◽

Scientific Community ◽

Xylella Fastidiosa ◽

Expression System ◽

Crop Protection ◽

Negative Bacterium ◽

In Planta ◽

Online Tool

The re-emergence of Gram-negative bacterium Xylella fastidiosa in Europe in 2013 impelled the scientific community to discover novel strategies for crop protection. The wide host range of Xylella indicates the existence of yet not characterized pathogenic mechanisms to overcome plant defenses. The recent uprising accuracy of a variety of bioinformatics tools, with the ability to predict the function of putative microbial protein represent a useful approach for understanding which of these proteins are associated with pathogens virulence. In this study we collected a number of putative effectors from two X. fastidiosa strains: Temecula1 and CoDiRo and the subspecies (ssp.) Sandyi Ann-1. We designed an in-planta Agrobacterium based expression system that drives the expressed proteins to the cell apoplast, in order to investigate their ability to activate defense in various model plants. Furthermore, we organized the resulted proteins according to their sequential and structural similarities via the I-TASSER online tool. We identified that various X. fastidiosa proteins were able to differentially elicit cell death-like phenotypes in Nicotiana tabacum, N. sylvestris and N. benthamiana. These proteins are members of different enzymatic groups: a) hydrolases/hydrolases inhibitors, b) serine proteases and c) metal transferases. Collectively, we identified structurally similar proteins that were able to differentially elicit cell death-like phenotypes in different cultivars of the same species. Our findings provide the bases for further studies on the mechanisms that underlie host-defense activation by X. fastidiosa putative effectors, as well as, pathogens adaptation in susceptible hosts.

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Predication of the Effector Proteins Secreted by Fusarium sacchari Using Genomic Analysis and Heterogenous Expression

Journal of Fungi ◽

10.3390/jof8010059 ◽

2022 ◽

Vol 8 (1) ◽

pp. 59

Author(s):

Zhen Huang ◽

Huixue Li ◽

Yuming Zhou ◽

Yixue Bao ◽

Zhenzhen Duan ◽

...

Keyword(s):

Cell Death ◽

Fungal Infections ◽

Expression Profiles ◽

Expression System ◽

Pathogenic Fungi ◽

Effector Proteins ◽

Economic Losses ◽

Effector Molecules ◽

Pathogen Effector ◽

Fusarium Sacchari

One of the causative agents of pokkah boeng disease (PBD), which affects sugarcane crops globally, is the fungus Fusarium sacchari. These fungal infections reduce sugar quality and yield, resulting in severe economic losses. Effector proteins play important roles in the interactions between pathogenic fungi and plants. Here, we used bioinformatic prediction approaches to identify 316 candidate secreted effector proteins (CSEPs) in the complete genome of F. sacchari. In total, 95 CSEPs contained known conserved structures, representing 40 superfamilies and 18 domains, while an additional 91 CSEPs contained seven known motifs. Of the 130 CSEPs containing no known domains or motifs, 14 contained one of four novel motifs. A heterogeneous expression system in Nicotiana benthamiana was used to investigate the functions of 163 CSEPs. Seven CSEPs suppressed BAX-triggered programmed cell death in N. benthamiana, while four caused cell death in N. benthamiana. The expression profiles of these eleven CSEPs during F. sacchari infection suggested that they may be involved in sugarcane-F. sacchari interaction. Our results establish a basis for further studies of the role of effector molecules in pathogen–sugarcane interactions, and provide a framework for future predictions of pathogen effector molecules.

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The Effect of Fusarium verticillioides Fumonisins on Fatty Acids, Sphingolipids, and Oxylipins in Maize Germlings

International Journal of Molecular Sciences ◽

10.3390/ijms22052435 ◽

2021 ◽

Vol 22 (5) ◽

pp. 2435

Author(s):

Marzia Beccaccioli ◽

Manuel Salustri ◽

Valeria Scala ◽

Matteo Ludovici ◽

Andrea Cacciotti ◽

...

Keyword(s):

Fatty Acids ◽

Fusarium Verticillioides ◽

Fungal Growth ◽

Defense Responses ◽

Ceramide Synthase ◽

Ear Rot ◽

In Planta ◽

Disease Symptoms ◽

The Impact

Fusarium verticillioides causes multiple diseases of Zea mays (maize) including ear and seedling rots, contaminates seeds and seed products worldwide with toxic chemicals called fumonisins. The role of fumonisins in disease is unclear because, although they are not required for ear rot, they are required for seedling diseases. Disease symptoms may be due to the ability of fumonisins to inhibit ceramide synthase activity, the expected cause of lipids (fatty acids, oxylipins, and sphingolipids) alteration in infected plants. In this study, we explored the impact of fumonisins on fatty acid, oxylipin, and sphingolipid levels in planta and how these changes affect F. verticillioides growth in maize. The identity and levels of principal fatty acids, oxylipins, and over 50 sphingolipids were evaluated by chromatography followed by mass spectrometry in maize infected with an F. verticillioides fumonisin-producing wild-type strain and a fumonisin-deficient mutant, after different periods of growth. Plant hormones associated with defense responses, i.e., salicylic and jasmonic acid, were also evaluated. We suggest that fumonisins produced by F. verticillioides alter maize lipid metabolism, which help switch fungal growth from a relatively harmless endophyte to a destructive necrotroph.

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The Ustilago hordei–Barley Interaction is a Versatile System for Characterization of Fungal Effectors

Journal of Fungi ◽

10.3390/jof7020086 ◽

2021 ◽

Vol 7 (2) ◽

pp. 86

Author(s):

Bilal Ökmen ◽

Daniela Schwammbach ◽

Guus Bakkeren ◽

Ulla Neumann ◽

Gunther Doehlemann

Keyword(s):

Fungal Pathogens ◽

Plant Pathogens ◽

Expression System ◽

Pathogenic Fungi ◽

Effector Proteins ◽

Mating Partner ◽

Fungal Virulence ◽

Functional Studies ◽

Infection Structures ◽

Ustilago Hordei

Obligate biotrophic fungal pathogens, such as Blumeria graminis and Puccinia graminis, are amongst the most devastating plant pathogens, causing dramatic yield losses in many economically important crops worldwide. However, a lack of reliable tools for the efficient genetic transformation has hampered studies into the molecular basis of their virulence or pathogenicity. In this study, we present the Ustilago hordei–barley pathosystem as a model to characterize effectors from different plant pathogenic fungi. We generate U. hordei solopathogenic strains, which form infectious filaments without the presence of a compatible mating partner. Solopathogenic strains are suitable for heterologous expression system for fungal virulence factors. A highly efficient Crispr/Cas9 gene editing system is made available for U. hordei. In addition, U. hordei infection structures during barley colonization are analyzed using transmission electron microscopy, showing that U. hordei forms intracellular infection structures sharing high similarity to haustoria formed by obligate rust and powdery mildew fungi. Thus, U. hordei has high potential as a fungal expression platform for functional studies of heterologous effector proteins in barley.

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