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Functional Characterization of Extracellular Protease Inhibitors of Phytophthora Spp and Their Targets Tomato Proteases

Functional Characterization of Extracellular Protease Inhibitors of Phytophthora Spp and Their Targets Tomato Proteases
Author: Jing Song
Publisher:
Total Pages:
Release: 2007
Genre: Phytophthora
ISBN:

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Abstract: The interplay between proteases and protease inhibitors during plant-pathogen interaction represents a common strategy for defense and counter-defense. The plant pathogens Phytophthora infestans and Phytophthora mirabilis secrete effectors such as protease inhibitors that facilitate host colonization through a defense-counterdefense mechanism. The P. infestans serine protease inhibitors EPI1 and EPI10 physically bind and inhibit the tomato serine protease P69B. On the other hand, the P. infestans cysteine protease inhibitor EPIC2B targets PIP1, a papain-like protease that has close similarity to another tomato cysteine protease Rcr3, which is required for the fungal resistance and Avr2 hypersensitivity in Cf-2 tomato. The objective of this research is to characterize these protease inhibitors and their association with specific targets in the host. We studied the structure and activities of these protease inhibitors and their target proteases using recombinant proteins expressed in Escherichia coli and Nicotiana benthamiana. PIP1-His was pulled down using coimmunoprecipitation with anti-FLAG resin from N. benthamiana apoplast by recombinant protein FLAG-EPIC2B, suggesting physical interaction of EPIC2B and PIP1. Similarly, tomato protease Rcr3pim was shown to be a common target for both the Cladosporium fulvum effector Avr2 and P. infestans effector EPIC2B using pull down assays and DCG-04 activity profiling. However, unlike Avr2, EPIC2B is a reversible inhibitor of Rcr3pim and does not trigger hypersensitivity on Cf-2/Rcr3pim tomato. We also found that the rcr3-3 mutant of tomato that carries a premature stop codon in the Rcr3 gene exhibits enhanced susceptibility to P. infestans, suggesting a role for Rcr3pim in basal defense. It appears that relative to C. fulvum, P. infestans evolved a cunning effector that carries virulence activity without triggering plant innate immunity. Like EPIC1, PmEPIC1, an EPIC1 homolog from P. mirabilis, was also found to bind Rcr3pim but not PIP1. Unlike EPIC2B, neither EPIC1 nor PmEPIC1 binds or inhibits PIP1. This work was possible by the ability to express and purify proteins in the apoplast of N. benthamiana. Three tomato proteases with C-terminal 6XHistidine tag were successfully expressed in N. benthamiana apoplast. Our findings suggest that C-terminal His-tagging of proteins in N. benthamiana apoplast is efficient enough to enable purification of functional proteins.


Functional Characterization of Extracellular Protease Inhibitors of Phytophthora Infestans

Functional Characterization of Extracellular Protease Inhibitors of Phytophthora Infestans
Author: Miaoying Tian
Publisher:
Total Pages:
Release: 2005
Genre: Late blight of potato
ISBN:

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Abstract: The oomycetes form one of several lineages within the eukaryotes that independently evolved a parasitic lifestyle and are thought to have developed unique mechanisms of pathogenicity. The devastating oomycete plant pathogen Phytophthora infestans causes late blight, a ravaging disease of potato and tomato. Little is known about processes associated with P. infestans pathogenesis, particularly the suppression of host defense responses. We used data mining of P. infestans sequence databases to identify 18 extracellular protease inhibitors belonging to two major structural classes: (i) Kazal-like serine protease inhibitors (EPI1 to EPI14) and (ii) cystatin-like cysteine protease inhibitors (EPIC1 to EPIC4). A variety of molecular, biochemical and bioinformatic approaches were employed to functionally characterize these genes and investigate their roles in pathogen virulence. The 14 EPI proteins form a diverse family and appear to have evolved by domain shuffling, gene duplication, and diversifying selection to target a diverse array of serine proteases. Recombinant EPI1 and EPI10 proteins inhibited subtilisin A among major serine proteases, and inhibited and interacted with tomato P69B subtilase, a pathogenesis-related protein belonging to PR7 class. The recombinant cystatin-like cysteine protease inhibitor EPIC2B interacted with a novel tomato papain-like extracellular cysteine protease PIP1 with an implicated role in plant defense. PIP1 is closely related to Rcr3, an apoplastic cysteine protease required for tomato Cf-2 and Cladosporium fulvm Avr2-dependent defense response. Both EPIC1 and EPIC2B interacted with Rcr3. Interactions with plant defense-related proteases suggest a counterdefense role of these extracellular protease inhibitors. Interestingly, EPIC1 and EPIC2B were degraded by tomato pathogenesis-related P69B subtilase and EPI1 protected both proteins from degradation, indicating that EPI1 contributes to virulence by protecting pathogen proteins from degradation by defense-related proteases. In addition, our overall results suggest that complex cascades of inhibition of host proteases by diverse extracellular protease inhibitors of P. infestans might occur in the plant apoplast during infection, thus leading to multifaceted suppression of plant defense responses. Both Kazal-like and cystatin-like inhibitors are widespread in the oomycetes, but have not been reported in other microbial plant pathogens. Inhibition of host proteases by P. infestans protease inhibitors is proposed to be a novel mechanism of pathogen suppression of plant defense.


Plant Proteases

Plant Proteases
Author: Mercedes Diaz-Mendoza
Publisher: Frontiers Media SA
Total Pages: 157
Release: 2020-01-24
Genre:
ISBN: 2889633993

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Plant proteases are involved in most aspects of plant physiology and development, playing key roles in the generation of signaling molecules and as regulators of essential cellular processes such as cell division and metabolism. They take part in important pathways like protein turnover by the degradation of misfolded proteins and the ubiquitin-proteasome pathway, and they are responsible for post-translational modifications of proteins by proteolysis at highly specific sites. Proteases are also implicated in a great variety of environmentally controlled processes, including mobilization of storage proteins during seed germination, development of seedlings, senescence, programmed cell death and defense mechanisms against pests and pathogens. However, in spite of their importance, little is known about the functions and mode of actions of specific plant proteases. This Research Topic collects contributions covering diverse aspects of plant proteases research.


How Can Secretomics Help Unravel the Secrets of Plant-Microbe Interactions?

How Can Secretomics Help Unravel the Secrets of Plant-Microbe Interactions?
Author: Delphine Vincent
Publisher: Frontiers Media SA
Total Pages: 190
Release: 2017-01-27
Genre:
ISBN: 2889450872

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Secretomics describes the global study of proteins that are secreted by a cell, a tissue or an organism, and has recently emerged as a field for which interest is rapidly growing. The term secretome was first coined at the turn of the millennium and was defined to comprise not only the native secreted proteins released into the extracellular space but also the components of machineries for protein secretion. Two secretory pathways have been described in fungi: i) the canonical pathway through which proteins bearing a N-terminal peptide signal can traverse the endoplasmic reticulum and Golgi apparatus, and ii) the unconventional pathway for proteins lacking a peptide signal. Protein secretion systems are more diverse in bacteria, in which types I to VII pathways as well as Sec or two-arginine (Tat) pathways have been described. In oomycete species, effectors are mostly small proteins containing an N-terminal signal peptide for secretion and additional C-terminal motifs such as RXLRs and CRNs for host targeting. It has recently been shown that oomycetes exploit non-conventional secretion mechanisms to transfer certain proteins to the extracellular environment. Other non-classical secretion systems involved in plant-fugal interaction include extracellular vesicles (EVs, Figure 1 from Samuel et al 2016 Front. Plant Sci. 6:766.). The versatility of oomycetes, fungi and bacteria allows them to associate with plants in many ways depending on whether they are biotroph, hemibiotroph, necrotroph, or saprotroph. When interacting with a live organism, a microbe will invade its plant host and manipulate its metabolisms either detrimentally if it is a pathogen or beneficially if it is a symbiote. Deciphering secretomes became a crucial biological question when an increasing body of evidence indicated that secreted proteins were the main effectors initiating interactions, whether of pathogenic or symbiotic nature, between microbes and their plant hosts. Secretomics may help to contribute to the global food security and to the ecosystem sustainability by addressing issues in i) plant biosecurity, with the design of crops resistant to pathogens, ii) crop yield enhancement, for example driven by arbuscular mycorrhizal fungi helping plant hosts utilise phosphate from the soil hence increase biomass, and iii) renewable energy, through the identification of microbial enzymes able to augment the bio-conversion of plant lignocellulosic materials for the production of second generation biofuels that do not compete with food production. To this day, more than a hundred secretomics studies have been published on all taxa and the number of publications is increasing steadily. Secretory pathways have been described in various species of microbes and/or their plant hosts, yet the functions of proteins secreted outside the cell remain to be fully grasped. This Research Topic aims at discussing how secretomics can assist the scientists in gaining knowledge about the mechanisms underpinning plant-microbe interactions.


Functional Genomics

Functional Genomics
Author: Michael J. Brownstein
Publisher: Springer Science & Business Media
Total Pages: 264
Release: 2008-02-03
Genre: Science
ISBN: 159259364X

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This collection of robust, readily reproducible methods for microarray-based studies includes expert guidance in the optimal data analysis and informatics. On the methods side are proven techniques for monitoring subcellular RNA localization en masse, for mapping chromosomes at the resolution of a single gene, and for surveying the steady-state genome-wide distribution of DNA binding proteins in vivo. For those workers dealing with massive data sets, the book discusses the methodological aspects of data analysis and informatics in the design of microarray experiments, the choice of test statistic, and the assessment of observational significance, data reduction, and clustering.


Plant Relationships

Plant Relationships
Author: Holger B. Deising
Publisher: Springer Science & Business Media
Total Pages: 408
Release: 2009-02-07
Genre: Science
ISBN: 3540874070

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Since the publication of the first edition of "The Mycota Vol. V – Plant Relationships" in 1997, tremendous advances in fungal molecular biology and biochemistry have taken place; and both light and electron microscopical techniques have improved considerably. These new insights led to a better understanding of the relationships between fungi and plants; and a completely revised new edition of Plant Relationships could be produced, providing an up-to-date overview on mutualistic and pathogenic interactions. In 18 chapters internationally acknowledged authors present reviews on fungal lifestyles, mechanisms of their interactions with their host plants, signal perception and transduction, and plant defense responses directed against attack by fungal pathogens. Highlighting the recent developments in fungus-plant interactions, this volume is indispensable for researchers, lecturers and students in microbiology, mycology and plant sciences, including plant pathology.


Annual Plant Reviews, Plant-Pathogen Interactions

Annual Plant Reviews, Plant-Pathogen Interactions
Author: Nicholas J. Talbot
Publisher: John Wiley & Sons
Total Pages: 264
Release: 2009-02-12
Genre: Science
ISBN: 1405147938

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Annual Plant Reviews, Volume 11 Plant diseases are destructive and threaten virtually any cropgrown on a commercial scale. They are kept in check by plantbreeding strategies that have introgressed disease resistance genesinto many important crops, and by the deployment of costly controlmeasures, such as antibiotics and fungicides. However, the capacityfor the agents of plant disease – viruses, bacteria, fungiand oomycetes – to adapt to new conditions, overcomingdisease resistance and becoming resistant to pesticides, is verygreat. For these reasons, understanding the biology of plantdiseases is essential for the development of durable controlstrategies. This volume provides an overview of our current knowledge ofplant-pathogen interactions and the establishment of plant disease,drawing together fundamental new information on plant infectionmechanisms and host responses. The role of molecular signals, generegulation and the physiology of pathogenic organisms areemphasised, but the role of the prevailing environment in theconditioning of disease is also discussed. This is a book for researchers and professionals in plantpathology, cell biology, molecular biology and genetics.


Microbial Enzymes: Roles and Applications in Industries

Microbial Enzymes: Roles and Applications in Industries
Author: Naveen Kumar Arora
Publisher: Springer Nature
Total Pages: 331
Release: 2020-04-28
Genre: Science
ISBN: 981151710X

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“Microbial Enzymes: Roles and applications in industry” offers an essential update on the field of microbial biotechnology, and presents the latest information on a range of microbial enzymes such as fructosyltransferase, laccases, amylases, lipase, and cholesterol oxidase, as well as their potential applications in various industries. Production and optimisation technologies for several industrially relevant microbial enzymes are also addressed. In recent years, genetic engineering has opened up new possibilities for redesigning microbial enzymes that are useful in multiple industries, an aspect that the book explores. In addition, it demonstrates how some of the emerging issues in the fields of agriculture, environment and human health can be resolved with the aid of green technologies based on microbial enzymes. The topics covered here will not only provide a better understanding of the commercial applications of microbial enzymes, but also outline futuristic approaches to use microbial enzymes as driver of industrial sustainability. Lastly, the book is intended to provide readers with an overview of recent applications of microbial enzymes in various industrial sectors, and to pique researchers’ interest in the development of novel microbial enzyme technologies to meet the changing needs of industry.


Effectors in Plant-Microbe Interactions

Effectors in Plant-Microbe Interactions
Author: Francis Martin
Publisher: John Wiley & Sons
Total Pages: 442
Release: 2012-01-03
Genre: Science
ISBN: 0470958227

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Plants and microbes interact in a complex relationship that can have both harmful and beneficial impacts on both plant and microbial communities. Effectors, secreted microbial molecules that alter plant processes and facilitate colonization, are central to understanding the complicated interplay between plants and microbes. Effectors in Plant-Microbe Interactions unlocks the molecular basis of this important class of microbial molecules and describes their diverse and complex interactions with host plants. Effectors in Plant Microbe Interactions is divided into five sections that take stock of the current knowledge on effectors of plant-associated organisms. Coverage ranges from the impact of bacterial, fungal and oomycete effectors on plant immunity and high-throughput genomic analysis of effectors to the function and trafficking of these microbial molecules. The final section looks at effectors secreted by other eukaryotic microbes that are the focus of current and future research efforts. Written by leading international experts in plant-microbe interactions, Effectors in Plant Microbe Interactions, will be an essential volume for plant biologists, microbiologists, pathologists, and geneticists.