Reverse Genetics for Functional Genomics of Phytopathogenic Fungi and Oomycetes

Sequencing of over 40 fungal and oomycete genomes has been completed. The next major challenge in modern fungal/oomycete biology is now to translate this plethora of genome sequence information into biological functions. Reverse genetics has emerged as a seminal tool for functional genomics investigations. Techniques utilized for reverse genetics like targeted gene disruption/replacement, gene silencing, insertional mutagenesis, and targeting induced local lesions in genomes will contribute greatly to the understanding of gene function of fungal and oomycete pathogens. This paper provides an overview on high-throughput reverse genetics approaches to decode fungal/oomycete genomes

Clubroot resistance gene Rcr6 in Brassica nigra resides in a genomic region homologous to chromosome A08 in B. rapa

Background: Clubroot, caused by Plasmodiophora brassicae Woronin, is a very important disease of Brassica species. Management of clubroot relies heavily on genetic resistance. In a cross of Brassica nigra lines PI 219576 (highly resistant, R) × CR2748 (highly susceptible, S) to clubroot, all F1 plants were resistant to clubroot. There was a 1:1 ratio of R:S in the BC1 and 3R:1S in the F2, which indicated that a single dominant gene controlled clubroot resistance in PI 219576. This gene was designated Rcr6. Mapping of Rcr6 was performed using genome sequencing information from A-genome of B. rapa and B-genome of B. nigra though bulked segregant RNA sequencing (BSR-Seq) and further mapping with Kompetitive Allele Specific PCR (KASP) analysis. Results: Reads of R and S bulks from BSR-Seq were initially aligned onto B. rapa (A-genome; B. nigra has the B-genome) where Rcr6 was associated with chromosome A08. KASP analysis showed that Rcr6 was flanked by SNP markers homologous to the region of 14.8-15.4 Mb of chromosome A08. There were 190 genes annotated in this region, with five genes (Bra010552, Bra010588, Bra010589, Bra010590 and Bra010663) identified as encoding the toll-interleukin-1 receptor / nucleotide-binding site / leucine-rich-repeat (TIR-NBS-LRR; TNL) class of proteins. The reads from BSR-Seq were then aligned into a draft B-genome of B. nigra, where Rcr6 was mapped on chromosome B3. KASP analysis indicated that Rcr6 was located on chromosome B3 in a 0.5 Mb region from 6.1-6.6 Mb. Only one TNL gene homologous to the B. rapa gene Bra010663 was identified in the target region. This gene is a likely candidate for Rcr6. Subsequent analysis of the Rcr6 equivalent region based on a published B. nigra genome was performed. This gene is located into chromosome B7 of the published B-genome, homologous to BniB015819. Conclusion: Rcr6 was the first gene identified and mapped in the B-genome of Brassica species. It resides in a genomic region homologous to chromosome A08 of A-genome. Based on this finding, it could possibly integrate into A08 of B. napus using marker assisted selection with SNP markers tightly linked to Rcr6 developed in this study

Peroxisomal Alanine: Glyoxylate Aminotransferase AGT1 Is Indispensable for Appressorium Function of the Rice Blast Pathogen, Magnaporthe oryzae

The role of β-oxidation and the glyoxylate cycle in fungal pathogenesis is well documented. However, an ambiguity still remains over their interaction in peroxisomes to facilitate fungal pathogenicity and virulence. In this report, we characterize a gene encoding an alanine, glyoxylate aminotransferase 1 (AGT1) in Magnaporthe oryzae, the causative agent of rice blast disease, and demonstrate that AGT1 is required for pathogenicity of M. oryzae. Targeted deletion of AGT1 resulted in the failure of penetration via appressoria; therefore, mutants lacking the gene were unable to induce blast symptoms on the hosts rice and barley. This penetration failure may be associated with a disruption in lipid mobilization during conidial germination as turgor generation in the appressorium requires mobilization of lipid reserves from the conidium. Analysis of enhanced green fluorescent protein expression using the transcriptional and translational fusion with the AGT1 promoter and open reading frame, respectively, revealed that AGT1 expressed constitutively in all in vitro grown cell types and during in planta colonization, and localized in peroxisomes. Peroxisomal localization was further confirmed by colocalization with red fluorescent protein fused with the peroxisomal targeting signal 1. Surprisingly, conidia produced by the Δagt1 mutant were unable to form appressoria on artificial inductive surfaces, even after prolonged incubation. When supplemented with nicotinamide adenine dinucleotide (NAD+)+pyruvate, appressorium formation was restored on an artificial inductive surface. Taken together, our data indicate that AGT1-dependent pyruvate formation by transferring an amino group of alanine to glyoxylate, an intermediate of the glyoxylate cycle is required for lipid mobilization and utilization. This pyruvate can be converted to non-fermentable carbon sources, which may require reoxidation of NADH generated by the β-oxidation of fatty acids to NAD+ in peroxisomes. Therefore, it may provide a means to maintain redox homeostasis in appressoria

New insights in to ancient resistance: the molecular side of cell wall appositions

The epidermis lies at the interface between a plant and its environment. As such, the epidermis is crucial for protecting the plant against environmental insults. We focus primarily on cell wall reinforcement-mediated penetration resistance (papilla-resistance) against fungal pathogen attack. The epidermal cell layer of cereal leaves is the only tissue interacting with the powdery mildew fungus, Blumeria graminis, and papilla formation at sites of fungal penetration attempts provides a basal resistance, hampering fungal invasion irrespective of host specific compatibility or incompatibility. To elucidate the genetic scaffolding of penetration resistance mechanisms, we constructed a cDNA library from wheat leaf epidermis at 24-48 h post inoculation with B. graminis f. sp. tritici. We have sequenced 3,000 expressed sequence tags (ESTs) from this cDNA library. EST analysis revealed a large proportion of genes involved in plant defense/stress responses (1/3) and a low frequency of “house-keeping” genes. Enrichment of defense genes from this EST collection has allowed us to identify several defense and signaling pathways that have been hitherto poorly characterized, including cell wall biosynthesis, vesicle trafficking, redox regulation and metal homeostasis. Our results suggest that a global analysis of transcripts from this epidermis-specific cDNA library makes it feasible to define a full set of genes involved in early plant resistance associated with cell wall modifications.L’épiderme se situe à l’interface entre la plante et son environnement. L’épiderme est donc essentiel à la protection de la plante contre les assauts de l’environnement. Nous nous sommes concentrés sur la résistance à la pénétration par l’intermédiaire du renforcement de la paroi cellulaire (résistance papillaire) contre les attaques de champignons pathogènes. La couche de cellules épidermiques des feuilles des céréales est le seul tissu qui interagit avec le champignon de l’oïdium, le Blumeria graminis, et la formation de papilles aux sites des tentatives de pénétration du champignon fournit une résistance de base, empêchant l’invasion fongique peu importe que l’hôte soit compatible ou incompatible. Afin d’élucider l’échafaudage génétique des mécanismes de résistance à la pénétration, nous avons construit une bibliothèque génomique à partir d’épiderme de feuille de blé recueilli 24 à 48 h après inoculation avec le B. graminis f. sp. tritici. Nous avons séquencé 3000 séquences EST à partir de cette bibliothèque. L’analyse des séquences EST a montré qu’il y avait une proportion importante de gènes impliqués dans la défense de la plante ou les réponses aux stress (1/3) et une faible teneur en gènes « d’intendance ». L’enrichissement en gènes de défense de cette collection de séquences EST nous a permis d’identifier plusieurs voies de défense et de signalisation qui ont été peu caractérisées jusqu’à présent, y compris la biosynthèse de la paroi cellulaire, le transport au niveau des vésicules, la régulation de l’oxydoréduction et l’homéostasie des métaux. Nos résultats laissent penser qu’une analyse globale des produits de transcription provenant de cette bibliothèque génomique spécifique à l’épiderme pourrait permettre la description d’un ensemble complet de gènes impliqués dans la résistance précoce des plantes associée aux modifications de la paroi cellulaire

Cataloging proteins putatively secreted during the biotrophy-necrotrophy transition of the anthracnose pathogen Colletotrichum truncatum

Hemibiotrophic phytopathogenic fungi cause devastating diseases in agronomically important crops. These fungal pathogens exploit a stealth bi-phasic infection strategy to colonize host plants. Their morphological and nutritional transition from biotrophy (characterized by voluminous intracellular primary hyphae) to necrotrophy (characterized by thin secondary hyphae) known as the biotrophy-necrotrophy switch (hemibiotrophy) is critical in symptom and disease development. To establish successful hemibiotrophic parasitism, pathogens likely secrete suites of proteins at the switch that constitute the biotrophy-necrotrophy switch secretome. To catalog such proteins, a directional cDNA library was constructed from mRNA isolated from infected Lens culinaris leaflet tissues displaying the switch of Colletotrichum truncatum, and 5,000 expressed sequence tags (ESTs) were generated. Four potential groups [hydrolytic enzymes, cell envelope-associated proteins (CEAPs), candidate effectors and proteins with diverse functions] were identified from pathogen-derived ESTs. Expression profiling of transcripts encoding CEAPs and candidate effectors in an infection time-course revealed that the majority of these transcripts were expressed or induced during the necrotrophic phase and repressed during the biotrophic phase of in planta colonization, indicating the massive accumulation of proteins at the switch. Taken together, our data suggest that the hemibiotrophic mode of fungal proliferation entails complex interactions of a pathogen with its host wherein the pathogen requires live host cells prior to switching to the necrotrophic phase. The microbial proteins employed during pathogenesis are likely to have defined roles at specific stages of pathogenesis

Overexpression of a novel biotrophy-specific colletotrichum truncatum Effector, CtNUDIX, in hemibiotrophic fungal phytopathogens causes incompatibility with their host plants

The hemibiotrophic fungus Colletotrichum truncatum causes anthracnose disease on lentils and a few other grain legumes. It shows initial symptomless intracellular growth, where colonized host cells remain viable (biotrophy), and then switches to necrotrophic growth, killing the colonized host plant tissues. Here, we report a novel effector gene, CtNUDIX, from C. truncatum that is exclusively expressed during the late biotrophic phase (before the switch to necrotrophy) and elicits a hypersensitive response (HR)-like cell death in tobacco leaves transiently expressing the effector. CtNUDIX homologs, which contain a signal peptide and a Nudix hydrolase domain, may be unique to hemibiotrophic fungal and fungus-like plant pathogens. CtNUDIX lacking a signal peptide or a Nudix motif failed to induce cell death in tobacco. Expression of CtNUDIX:eGFP in tobacco suggested that the fusion protein might act on the host cell plasma membrane. Overexpression of CtNUDIX in C. truncatum and the rice blast pathogen, Magnaporthe oryzae, resulted in incompatibility with the hosts lentil and barley, respectively, by causing an HR-like response in infected host cells associated with the biotrophic invasive hyphae. These results suggest that C. truncatum and possibly M. oryzae elicit cell death to signal the transition from biotrophy to necrotrophy.Peer reviewed: YesNRC publication: Ye

An important role for secreted esterase in disease establishment of the wheat powdery mildew fungus Blumeria graminis f. sp. tritici

Abstract: The activity of esterase secreted by conidia of wheat powdery mildew fungus, Blumeria graminis f. sp. tritici, was assayed using indoxyl acetate hydrolysis, which generates indigo blue crystals. Mature, ungerminated, and germinating conidia secrete esterase(s) on artificial media and on plant leaf surfaces. The activity of these esterases was inhibited by diisopropyl fluorophosphate, which is selective for serine esterases. When conidia were inoculated on wheat leaves pretreated with diisopropyl fluorophosphate, both appressorial germ tube differentiation and symptom development were significantly impaired, indicating an important role of secreted serine esterases in wheat powdery mildew disease establishment. Key words: serine esterase, cutinase, pathogenesis, powdery mildew, wheat. Résumé : L'activité estérase sécrétée par les conidies de Blumeria graminis f. sp. tritici, le champignon responsable de l'oïdium (blanc) du blé, a été mesurée par l'hydrolyse de l'indoxyle acétate qui génère des cristaux bleu indigo. Les conidies matures, non germées, de même que les conidies en germination sécrètent des estérases sur du milieu artificiel et à la surface des feuilles des végétaux. L'activité de ces estérases était inhibée par le diisopropyle fluorophosphate, lequel est sélectif aux sérine estérases. Lorsque les conidies étaient inoculées sur les feuilles de blé prétraitées au diisopropyle fluorophosphate, la différenciation des tubes germinaux des appressoriums et le développement des symptômes étaient significativement réduits, ce qui indique que les sérine estérases secrétées jouent un rôle important dans l'établissement de l'oïdium chez le blé