Genome Editing and Protoplast Regeneration to Study Plant–Pathogen Interactions in the Model Plant Nicotiana benthamiana

Biotic diseases cause substantial agricultural losses annually, spurring research into plant pathogens and strategies to mitigate them. Nicotiana benthamiana is a commonly used model plant for studying plant–pathogen interactions because it is host to numerous plant pathogens and because many research tools are available for this species. The clustered regularly interspaced short palindromic repeats (CRISPR) system is one of several powerful tools available for targeted gene editing, a crucial strategy for analyzing gene function. Here, we demonstrate the use of various CRISPR-associated (Cas) proteins for gene editing of N. benthamiana protoplasts, including Staphylococcus aureus Cas9 (SaCas9), Streptococcus pyogenes Cas9 (SpCas9), Francisella novicida Cas12a (FnCas12a), and nCas9-activation-induced cytidine deaminase (nCas9-Target-AID). We successfully mutated Phytoene Desaturase (PDS) and Ethylene Receptor 1 (ETR1) and the disease-associated genes RNA-Dependent RNA Polymerase 6 (RDR6), and Suppressor of Gene Silencing 3 (SGS3), and confirmed that the mutated alleles were transmitted to progeny. sgs3 mutants showed the expected phenotype, including absence of trans-acting siRNA3 (TAS3) siRNA and abundant expression of the GFP reporter. Progeny of both sgs3 and rdr6 null mutants were sterile. Our analysis of the phenotypes of the regenerated progeny indicated that except for the predicted phenotypes, they grew normally, with no unexpected traits. These results confirmed the utility of gene editing followed by protoplast regeneration in N. benthamiana. We also developed a method for in vitro flowering and seed production in N. benthamiana, allowing the regenerants to produce progeny in vitro without environmental constraints.

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Nicotiana benthamiana: Its History and Future as a Model for Plant–Pathogen Interactions

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-00-00-1015-rev.testissue ◽

2015 ◽

Vol 2015 (1) ◽

pp. 28-39 ◽

Cited By ~ 11

Author(s):

Michael M. Goodin ◽

David Zaitlin ◽

Rayapati A. Naidu ◽

Steven A. Lommel

Keyword(s):

Plant Pathogen ◽

Nicotiana Benthamiana ◽

Plant Pathogen Interactions

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Co-Occurrence of Viruses, Plant Pathogens, and Symbionts in an Underexplored Hemipteran Clade

Frontiers in Cellular and Infection Microbiology ◽

10.3389/fcimb.2021.715998 ◽

2021 ◽

Vol 11 ◽

Author(s):

McKinlee M. Salazar ◽

Mônica T. Pupo ◽

Amanda M. V. Brown

Keyword(s):

Plant Pathogen ◽

Plant Pathogens ◽

Plant Viruses ◽

Metagenomic Sequencing ◽

Base Pairs ◽

Shotgun Metagenomic Sequencing ◽

Data Clusters ◽

Blast Database ◽

Insect Symbionts ◽

Plant Pathogen Interactions

Interactions between insect symbionts and plant pathogens are dynamic and complex, sometimes involving direct antagonism or synergy and sometimes involving ecological and evolutionary leaps, as insect symbionts transmit through plant tissues or plant pathogens transition to become insect symbionts. Hemipterans such as aphids, whiteflies, psyllids, leafhoppers, and planthoppers are well-studied plant pests that host diverse symbionts and vector plant pathogens. The related hemipteran treehoppers (family Membracidae) are less well-studied but offer a potentially new and diverse array of symbionts and plant pathogenic interactions through their distinct woody plant hosts and ecological interactions with diverse tending hymenopteran taxa. To explore membracid symbiont–pathogen diversity and co-occurrence, this study performed shotgun metagenomic sequencing on 20 samples (16 species) of treehopper, and characterized putative symbionts and pathogens using a combination of rapid blast database searches and phylogenetic analysis of assembled scaffolds and correlation analysis. Among the 8.7 billion base pairs of scaffolds assembled were matches to 9 potential plant pathogens, 12 potential primary and secondary insect endosymbionts, numerous bacteriophages, and other viruses, entomopathogens, and fungi. Notable discoveries include a divergent Brenneria plant pathogen-like organism, several bee-like Bombella and Asaia strains, novel strains of Arsenophonus-like and Sodalis-like symbionts, Ralstonia sp. and Ralstonia-type phages, Serratia sp., and APSE-type phages and bracoviruses. There were several short Phytoplasma and Spiroplasma matches, but there was no indication of plant viruses in these data. Clusters of positively correlated microbes such as yeast-like symbionts and Ralstonia, viruses and Serratia, and APSE phage with parasitoid-type bracoviruses suggest directions for future analyses. Together, results indicate membracids offer a rich palette for future study of symbiont–plant pathogen interactions.

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Structural basis of pathogen recognition by an integrated HMA domain in a plant NLR immune receptor

eLife ◽

10.7554/elife.08709 ◽

2015 ◽

Vol 4 ◽

Cited By ~ 102

Author(s):

A Maqbool ◽

H Saitoh ◽

M Franceschetti ◽

CEM Stevenson ◽

A Uemura ◽

...

Keyword(s):

Nicotiana Benthamiana ◽

Rice Blast ◽

Plant Pathogens ◽

Rice Cultivar ◽

Blast Disease ◽

Structural Basis ◽

Immune Receptor ◽

Immune Receptors ◽

Direct Recognition

Plants have evolved intracellular immune receptors to detect pathogen proteins known as effectors. How these immune receptors detect effectors remains poorly understood. Here we describe the structural basis for direct recognition of AVR-Pik, an effector from the rice blast pathogen, by the rice intracellular NLR immune receptor Pik. AVR-PikD binds a dimer of the Pikp-1 HMA integrated domain with nanomolar affinity. The crystal structure of the Pikp-HMA/AVR-PikD complex enabled design of mutations to alter protein interaction in yeast and in vitro, and perturb effector-mediated response both in a rice cultivar containing Pikp and upon expression of AVR-PikD and Pikp in the model plant Nicotiana benthamiana. These data reveal the molecular details of a recognition event, mediated by a novel integrated domain in an NLR, which initiates a plant immune response and resistance to rice blast disease. Such studies underpin novel opportunities for engineering disease resistance to plant pathogens in staple food crops.

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Apoplastic Cell Death-Inducing Proteins of Filamentous Plant Pathogens: Roles in Plant-Pathogen Interactions

Frontiers in Genetics ◽

10.3389/fgene.2020.00661 ◽

2020 ◽

Vol 11 ◽

Author(s):

Ya Li ◽

Yijuan Han ◽

Mengyu Qu ◽

Jia Chen ◽

Xiaofeng Chen ◽

...

Keyword(s):

Cell Death ◽

Plant Pathogen ◽

Plant Pathogens ◽

Plant Pathogen Interactions

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CRISPR-Cas systems in the plant pathogen Xanthomonas spp. and their impact on genome plasticity

10.1101/731166 ◽

2019 ◽

Author(s):

Paula Maria Moreira Martins ◽

Andre da Silva Xavier ◽

Marco Aurelio Takita ◽

Poliane Alfemas-Zerbini ◽

Alessandra Alves de Souza

Keyword(s):

Phylogenetic Analysis ◽

Plant Pathogen ◽

Significant Variation ◽

Crop Production ◽

Plant Pathogens ◽

Economic Losses ◽

Genome Plasticity ◽

Plant Pathogen Interactions ◽

Cas Proteins

AbstractXanthomonas is one of the most important bacterial genera of plant pathogens causing economic losses in crop production worldwide. Despite its importance, many aspects of basic Xanthomonas biology remain unknown or understudied. Here, we present the first genus-wide analysis of CRISPR-Cas in Xanthomonas and describe specific aspects of its occurrence. Our results show that Xanthomonas genomes harbour subtype I-C and I-F CRISPR-Cas systems and that species belonging to distantly Xanthomonas-related genera in Xanthomonadaceae exhibit the same configuration of coexistence of the I-C and I-F CRISPR subtypes. Additionally, phylogenetic analysis using Cas proteins indicated that the CRISPR systems present in Xanthomonas spp. are the result of an ancient acquisition. Despite the close phylogeny of these systems, they present significant variation in both the number and targets of spacers. An interesting characteristic observed in this study was that the identified plasmid-targeting spacers were always driven toward plasmids found in other Xanthomonas strains, indicating that CRISPR-Cas systems could be very effective in coping with plasmidial infections. Since many effectors are plasmid encoded, CRISPR-Cas might be driving specific characteristics of plant-pathogen interactions.

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Nicotiana benthamiana: Its History and Future as a Model for Plant–Pathogen Interactions

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-00-00-1015-rev.test ◽

2014 ◽

pp. MPMI-00-00-1015

Author(s):

Michael M. Goodin ◽

David Zaitlin ◽

Rayapati A. Naidu ◽

Steven A. Lommel

Keyword(s):

Plant Pathogen ◽

Nicotiana Benthamiana ◽

Plant Pathogen Interactions

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Functional Variation of Plant–Pathogen Interactions: New Concept and Methods for Virulence Data Analyses

Phytopathology ◽

10.1094/phyto-02-19-0041-le ◽

2019 ◽

Vol 109 (8) ◽

pp. 1324-1330 ◽

Cited By ~ 4

Author(s):

E. Kosman ◽

X. Chen ◽

A. Dreiseitl ◽

B. McCallum ◽

A. Lebeda ◽

...

Keyword(s):

Plant Pathogen ◽

Plant Pathogens ◽

Infection Type ◽

Lesion Size ◽

Data Sets ◽

Functional Variation ◽

Multiple Loci ◽

Plant Pathogen Interaction ◽

Few Data ◽

Plant Pathogen Interactions

Classical virulence analysis is based on discovering virulence phenotypes of isolates with regard to a composition of resistance genes in a differential set of host genotypes. With such a vision, virulence phenotypes are usually treated in a genetic manner as one of two possible alleles, either virulence or avirulence in a binary locus. Therefore, population genetics metrics and methods have become prevailing tools for analyzing virulence data at multiple loci. However, a basis for resolving binary virulence phenotypes is infection type (IT) data of host–pathogen interaction that express functional traits of each specific isolate in a given situation (particular host, environmental conditions, cultivation practice, and so on). IT is determined by symptoms and signs observed (e.g., lesion type, lesion size, coverage of leaf or leaf segments by mycelium, spore production and so on), and assessed by IT scores at a generally accepted scale for each plant–pathogen system. Thus, multiple IT profiles of isolates are obtained and can be subjected to analysis of functional variation within and among operational units of a pathogen. Such an approach may allow better utilization of the information available in the raw data, and reveal a functional (e.g., environmental) component of pathogen variation in addition to the genetic one. New methods for measuring functional variation of plant–pathogen interaction with IT data were developed. The methods need an appropriate assessment scale and expert estimations of dissimilarity between IT scores for each plant–pathogen system (an example is presented). Analyses of a few data sets at different hierarchical levels demonstrated discrepancies in results obtained with IT phenotypes versus binary virulence phenotypes. The ability to measure functional IT-based variation offers promise as an effective tool in the study of epidemics caused by plant pathogens.

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Trick or Treat: Microbial Pathogens Evolved Apoplastic Effectors Modulating Plant Susceptibility to Infection

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-07-17-0177-fi ◽

2018 ◽

Vol 31 (1) ◽

pp. 6-12 ◽

Cited By ~ 29

Author(s):

Yan Wang ◽

Yuanchao Wang

Keyword(s):

Immune Responses ◽

Plant Pathogen ◽

Plant Pathogens ◽

Plant Cell Wall ◽

Effector Proteins ◽

Microbial Pathogens ◽

Susceptibility To Infection ◽

Plant Pathogen Interactions ◽

Harsh Conditions ◽

Plant Susceptibility

The apoplastic space between the plant cell wall and the plasma membrane constitutes a major battleground for plant-pathogen interactions. To survive in harsh conditions in the plant apoplast, pathogens must cope with various immune responses. During infection, plant pathogens secrete an arsenal of effector proteins into the apoplast milieu, some of which are detected by the plant surveillance system and, thus, activate plant innate immunity. Effectors that evade plant perception act in modulating plant apoplast immunity to favor successful pathogen infection. The concerted actions of apoplastic effectors often determine the outcomes of plant-pathogen interactions. In this review, we summarize current advances on the understanding of apoplastic effectors and highlight the strategies employed by pathogens to counter host apoplastic defense.

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