VEL VETLEAF - COLLETOTRICHUM COCCODES PATHOSYSTEM: MOLECULAR MONITORING OF THE PATHOGEN AND GENE EXPRESSION ANALYSIS DURING PLANT PATHOGEN INTERACTION by Amélie L. Dauch A thesis submitted to the Graduate and Postdoctoral Studies Office in partial fulfillment of the requirements of the degree of Doctor of Science Department of Plant Science, McGiII University, Montreal Quebec, Canada © Amélie L. 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The author retains copyright L'auteur conserve la propriété du droit d'auteur ownership and moral rights in et des droits moraux qui protège cette thèse. this thesis. Neither the thesis Ni la thèse ni des extraits substantiels de nor substantial extracts from it celle-ci ne doivent être imprimés ou autrement may be printed or otherwise reproduits sans son autorisation. reproduced without the author's permission. ln compliance with the Canadian Conformément à la loi canadienne Privacy Act some supporting sur la protection de la vie privée, forms may have been removed quelques formulaires secondaires from this thesis. ont été enlevés de cette thèse. While these forms may be included Bien que ces formulaires in the document page count, aient inclus dans la pagination, their removal does not represent il n'y aura aucun contenu manquant. any loss of content from the thesis. ••• Canada ABSTRACT Colletotrichum coccodes strain DAOM 183088 is considered a potential bioherbicide for velvetleaf (Abutilon theophrasti), a devastating weed in North American corn and soybeans. Risk assessment studies have created a demand for an accurate and robust method to monitor this strain, and to distinguish it from indigenous background population of microorganisms present in the field. Safe biological control management of velvetleaf also requires comprehensive understanding of the pathogenicity determinants employed by this host-specifie fun gus to establish infection on velvetleaf, an aspect central to a safe biocontrol strategy task. In this study, molecular markers were designed that al10w strain specifie identification of the bioherbicide strain of C. coccodes and its identification within complex plant and soil matrices. An assay was developed to quantify C. coccodes from deliberate release field soil samples, in which biases caused by soil-originating PCR inhibitors were monitored on a sample per sample basis. The developed external control assay allowed for the estimation of target C. coccodes DNA quantities with normalization for the presence of PCR inhibitory compounds. Kinetic growth curves of disease development were performed for C. coccodes wild-type and T20-a (genetically engineered for hypervirulence with the NEP] (necrosis and ethylene inducing peptide) gene) strains on velvetleaf leaves over a period of 14 days after C. coccodes infection. The wild-type strain was more efficient at infecting velvetleaf than the transgenic T-20a strain, while expression of NEP] could not be detected suggesting that the introduced gene ii may not be transcriptionally active in the transformed strain, a result in conflict with previous observations. Velvetleaf and C. coccodes genes specifically up­ regulated at 12 and 24 h after fungal infection were cloned and differentially screened by microarrays. The resulting EST collection was sequenced and assigned to putative functions. Early gene up-regulation was confirmed by QRT­ PCR analysis for type 3 metallothionein, EREB, WRKY, and bZIP transcription factors, reticuline oxidase, ascorbate peroxidase, and ACC oxidase gene candidates. In addition, type 2, type 3 metallothionein, and bZIP gene expression profiles were investigated over a period of 14 days after C. coccodes infection, and the results indicated that C. coccodes altered the expression of aIl three gene analyzed. 111 RÉSUMÉ Le Colletotrichum coccodes (DAOM 183088) est un agent de lutte biologique (ALB) contre l'Abutilon (Abutilon theophrasti), une mauvaise herbe problématique pour les cultures de sOJa et de maïs en Amérique du Nord. L'évaluation des risques liés à la libération du C. coccodes en champs requiert un outil performant permettant le suivi environnemental de l' ALB. La compréhension des mécanismes de pathogénicité et de résistance est également un aspect crucial de la lutte biologique contre l'Abutilon. Des amorces de PCR spécifiques au seul isolat pathogène de l'Abutilon ont été développées et ont permis la détection du C. coccodes dans différents sols et plantes d'Abutilon. Un contrôle externe a également été élaboré de façon à pouvoir quantifier l' ALB dans des sols provenant de champs traités avec l' ALB tout en normalisant, pour chacun des échantillons analysés, les quantités d'ALB aux contenus en inhibiteurs de PCR. La croissance de 2 isolats de C. coccodes, sauvage et T-20a (transformé génétiquement avec le gène NEP 1 (necrosis and ethylene inducing peptide) à des fins de virulence accrue), a été suivie sur les feuilles d' Abutilon pendant 14 jours suivant l'infection des champignons. Le C. coccodes T-20a était moins virulent que le C. coccodes sauvage, et l'expression du gène NEP 1 n'a pu être détectée chez le T -20a, ce qui suggère que le transgène ne soit plus transcriptionnellement actif chez cet isolat. Les gènes du C. coccodes et de l' Abutilon sur-exprimés 12 et 24 h après l'infection du C. coccodes ont été clonés par SSH, analysés grâce à un criblage différentiel par biopuces, puis séquencés et leurs fonctions identifiées. La sur-expression des gènes de métallothioneine de type 3, facteurs de transcription iv EREB, WRKY, et bZIP, réticuline oxidase, ascorbate peroxidase, et ACC oxidase, 12 et 24 h après l'infection du C. coccodes, a été confinnée par PCR en temps réel. L'étude de l'expression des gènes de métallothioneine de types 2 et 3, et du facteur de transcription bZIP sur une période de 14 jours suivant l'infection du C. coccodes a également permis de démontrer que le champignon affectait l'expression de ces 3 gènes chez l'Abutilon. v ACKNOWLEDGMENTS 1 would like to express my gratitude and sincere acknowledgments to my supervisor, Dr. Suha Jabaji-Hare, whose expertise, understanding, and patience, added considerably to my graduate experience. 1 appreciate her knowledge and skills in many areas, and her assistance in writing reports (i.e., proposais, scholarship applications and this thesis) from which 1 preciously benefited. 1 would like to thank the other members of my committee, Dr. Alan Watson, Dr. Brian DriscoIl, and Dr. Marc Fortin for the assistance they provided at allievels of the research project. 1 would like to thank Dr. Philippe Seguin for taking time out from his busy schedule to help me with statistical analysis in chapters IV and V. Many thanks go to aIl Funlab fellows, in particular to Kui Wen, Danielle Morissette, Greg Taylor, Daniel Rivard, and Paula Cook, whose friendship and support greatly helped throughout these years. 1 would like to acknowledge Priscilla Barcial who provided plant and soil samples (chapter III), Sophie St-Louis for her technical assistance in soil sampling (chapter IV) and Dr. Byeongseok Ahn who performed C. coccodes velvetleaf disease severity trials (chapter V). The technical assistance of Paula Cook during chapter V experiments is greatly acknowledged. 1 thank Julie Beaulieu for her help in stand-alone blast analyses and Brian Driscoll for helping in the phylogeny analyses (chapter VI). Sincere acknowledgments go to Kui Wen for aIl the time vi she devoted to me, for her technical assistance in RNA extractions and statistical analyses. 1 must also acknowledge Dr. G. TeBeest (University of Arkansas, Arkansas, USA), Dr. G. Lazarovitz (SCFRC, AAFC, London, Ontario), and Dr. Carolyn E. Babcock (Canadian Collection of Fungal Cultures, AAFC, Ottawa, Ontario) who generously provided sorne of the Colletotrichum strains that have been used in chapter III. 1 recognize that this research would not have been possible without the financial assistance of FQRNT (Fonds québécois de la recherche sur la nature et les technologies)-IRDA (Institut de recherche et de développement en agroenvironnement) collaborative program (chapters III, IV; Dr Watson and Dr. Jabaji-Hare), Natural Sciences and Engineering Research Council of Canada (NSERC operating grant, chapter IV; NSERC biocontrol network grant, chapter IV and VI; NSERC discovery grant, chapter V and VI; Dr. Jabaji-Hare), the Department of Plant Science (teaching assistantships), the Canadian Phytopathological Society (travel awards), the Faculty of Agriculture and Environmental