Melanie Kalischuk

Tobacco Mosaic Virus Infection Results in an Increase in Recombination Frequency and Resistance to Viral, Bacterial, and Fungal Pathogens in the Progeny of Infected Tobacco Plants

Our previous experiments showed that infection of tobacco (Nicotiana tabacum) plants with Tobacco mosaic virus (TMV) leads to an increase in homologous recombination frequency (HRF). The progeny of infected plants also had an increased rate of rearrangements in resistance gene-like loci. Here, we report that tobacco plants infected with TMV exhibited an increase in HRF in two consecutive generations. Analysis of global genome methylation showed the hypermethylated genome in both generations of plants, whereas analysis of methylation via 5-methyl cytosine antibodies demonstrated both hypomethylation and hypermethylation. Analysis of the response of the progeny of infected plants to TMV, Pseudomonas syringae, or Phytophthora nicotianae revealed a significant delay in symptom development. Infection of these plants with TMV or P. syringae showed higher levels of induction of PATHOGENESIS-RELATED GENE1 gene expression and higher levels of callose deposition. Our experiments suggest that viral infection triggers specific changes in progeny that promote higher levels of HRF at the transgene and higher resistance to stress as compared with the progeny of unstressed plants. However, data reported in these studies do not establish evidence of a link between recombination frequency and stress resistance.

Genetic Composition of Phytophthora infestans in Canada Reveals Migration and Increased Diversity

A dramatic increase in the incidence of late blight and changes within populations of Phytophthora infestans have been observed in various regions of Canada. In this study, the occurrence of several new genotypes of the pathogen was documented with associated phenotypes that dominated pathogen populations. Genotype US-23, previously detected only among isolates from the United States, dominated in the western Canadian provinces of British Columbia, Alberta (AB), Saskatchewan, and Manitoba (MB). Although isolates of US-23 infect both potato and tomato, these isolates were the only genotype recovered from commercial garden centers in Canada. Isolates of genotype US-8, previously dominant throughout Canada, represented the only genotype detected from the eastern Canadian provinces of New Brunswick and Prince Edward Island. Isolates of other genotypes detected in Canada included US-11 in AB, US-24 in MB, and US-22 in Ontario (ON). An additional genotype was detected in ON which appears to be a derivative of US-22 that may have arisen through sexual reproduction. However, evidence of clonal reproduction dominated among the isolates collected, and opportunities for sexual reproduction were probably limited because of a surprising geographic separation of the A1 and A2 mating types in Canada. Sensitivity of the US-22, US-23, and US-24 isolates to the fungicide metalaxyl, movement of potato seed and transplants, and weather conditions may have contributed to reduced opportunities for contact between the mating types in fields in Canada. All P. infestans isolates were readily distinguished from other related oomycetes with RG57 restriction fragment length polymorphism analysis. Long-distance movement in seed tubers and garden center transplants may have contributed to the rapid spread of the P. infestans genotypes across Canada. Tracking pathogen movement and population composition should improve the ability to predict the genotypes expected each year in different regions of Canada.

Evolution and Management of the Irish Potato Famine Pathogen Phytophthora Infestans in Canada and the United States

Late blight, caused by Phytophthora infestans (Mont.) de Bary, is the most historically significant and economically destructive disease of potatoes (Solanum tuberosum L.). In addition to potato, P. infestans can also infect tomato and some other members of the Solanaceae, and this has contributed to the recent late blight epidemic in Canada and the United States. Propagation of P. infestans in Canada and the United States has been mainly through asexual reproduction and this has led to the development of several dominant clonal lineages. Various P. infestans markers have been developed that are invaluable in monitoring the evolution and movement of these P. infestans genotypes. Population diversity and disease incidence has increased through the development of systemic fungicide insensitivity and the transcontinental shipment of the pathogen on late blight infected potato tubers and tomato plantlets. Introduction of the P. infestans A2 mating type to several regions of Canada and the United States has also increased the opportunity for sexual reproduction and recombination, potentially contributing to greater P. infestans genetic diversity and pathogenicity. Advances in P. infestans molecular analysis have revealed a complex pathogen with a genome capable of evolving relatively quickly. Management of late blight will therefore require new, multifaceted strategies which include monitoring pathogen evolution and implementing sustainable production practices.ResumenEl tizón tardío, causado por Phytophthora infestans (Mont.) de Bary, es la enfermedad históricamente más significativa y económicamente destructiva de papa (Solanum tuberosum L.). Además de la papa, P. infestans también puede infectar tomate y a otros miembros de la familia Solanaceae, y esto ha contribuido a la reciente epidemia de tizón tardío en Canadá y los Estados Unidos. La propagación de P.infestans en estos dos países ha sido principalmente mediante reproducción asexual, lo que ha conducido al desarrollo de varias líneas clonales dominantes. Se han desarrollado varios marcadores para P. infestans que son invaluables en el seguimiento de la evolución y movimiento de estos genotipos de P. infestans. La diversidad de la población y la incidencia de la enfermedad han aumentado por vía del desarrollo de la insensibilidad a fungicidas sistémicos y del envío transcontinental del patógeno en tubérculos de papa infectados con tizón tardío y en plántulas de tomate. La introducción del tipo de compatibilidad A2 de P. infestans a varias regiones de Canadá y Estados Unidos también ha incrementado la oportunidad de reproducción sexual y recombinación, contribuyendo, potencialmente, a una mayor diversidad genética y patogenicidad de P. infestans. Avances en los análisis moleculares de P. infestans han revelado a un patógeno complejo con un genomio capaz de evolucionar relativamente rápido. El manejo del tizón tardío, entonces, requerirá de nuevas estrategias multifacéticas que incluyan monitoreo de la evolución del patógeno y la implementación de prácticas sustentables de producción.

Complete Genome Sequence of Phytopathogenic Pectobacterium atrosepticum Bacteriophage Peat1

ABSTRACT Pectobacterium atrosepticum is a common phytopathogen causing significant economic losses worldwide. To develop a biocontrol strategy for this blackleg pathogen of solanaceous plants, P. atrosepticum bacteriophage Peat1 was isolated and its genome completely sequenced. Interestingly, morphological and sequence analyses of the 45,633-bp genome revealed that phage Peat1 is a member of the family Podoviridae and most closely resembles the Klebsiella pneumoniae bacteriophage KP34. This is the first published complete genome sequence of a phytopathogenic P. atrosepticum bacteriophage, and details provide important information for the development of biocontrol by advancing our understanding of phage-phytopathogen interactions.

Priming with a double-stranded DNA virus alters Brassica rapa seed architecture and facilitates a defense response.

BACKGROUND Abiotic and biotic stresses alter genome stability and physiology of plants. Under some stressful situations, a state of stress tolerance can be passed on to the offspring rendering them more suitable to stressful events than their parents. In plants, the exploration of transgenerational response has remained exclusive to model species, such as Arabidopsis thaliana. Here, we expand transgenerational research to include Brassica rapa, a close relative to economically important plant canola (Brassica napus), as it is exposed to the biotic stress of a double-stranded DNA virus Cauliflower mosaic virus (CaMV). RESULTS Parent plants exposed to a low dose of 50ng purified CaMV virions just prior to the bolting stage produced significantly larger seeds than mock inoculated and healthy treatments. The progeny from these large seeds displayed resistance to the pathogen stress applied in the parental generation. Differences in defense pathways involving fatty acids, and primary and secondary metabolites were detected by de novo transcriptome sequencing of CaMV challenged progeny exhibiting different levels of resistance. CONCLUSIONS Our study highlights biological and cellular processes that may be linked to the growth and yield of economically important B. rapa, in a transgenerational manner. Although much remains unknown as to the mechanisms behind transgenerational inheritance, our work shows a disease resistance response that persists for several weeks and is associated with an increase in seed size. Evidence suggests that a number of changes involved in the persistent stress adaption are reflected in the transcriptome. The results from this study demonstrate that treating B. rapa with dsDNA virus within a critical time frame and with a specified amount of infectious pathogen produces economically important agricultural plants with superior coping strategies for growing in unfavorable conditions.

Characterization of Phytophthora infestans populations in Canada during 2012

Abstract The late blight pathogen (Phytophthora infestans) continues to cause major losses on potato and tomato in Canada and worldwide. An increased diversity of P. infestans and dramatic shifts in pathogen populations have occurred in Canada in recent years. In 2011, a survey identified different genotypes of P. infestans in Canada, including the new US-22, US-23 and US-24 genotypes, which were dominant in various Canadian provinces. In 2012, analysis of samples collected from infected potato and tomato plants from different regions in Canada indicated a rapid change in P. infestans populations in most provinces within a single year. For example, in Prince Edward Island, the US-8 genotype that dominated the P. infestans landscape for many years was displaced by the US-23 genotype, a phenomenon similar to that which occurred in western Canada in prior years. In British Columbia, however, US-11 and the new CA-12 were the dominant genotypes while in Ontario the tested isolates were US-22. Evidence for recombination was found, and increasing insensitivity to mefenoxam was apparent among isolates of some populations. Independent segregation of either Gpi, mating type or RG57 loci occurred in a number of the recombinant isolates, resulting in increased diversity of P. infestans populations. The unexpected change in composition of P. infestans genotypes supports the need for continued monitoring of this pathogen.

The Luteovirus P4 Movement Protein Is a Suppressor of Systemic RNA Silencing

The plant viral family Luteoviridae is divided into three genera: Luteovirus, Polerovirus and Enamovirus. Without assistance from another virus, members of the family are confined to the cells of the host plant’s vascular system. The first open reading frame (ORF) of poleroviruses and enamoviruses encodes P0 proteins which act as silencing suppressor proteins (VSRs) against the plant’s viral defense-mediating RNA silencing machinery. Luteoviruses, such as barley yellow dwarf virus-PAV (BYDV-PAV), however, have no P0 to carry out the VSR role, so we investigated whether other proteins or RNAs encoded by BYDV-PAV confer protection against the plant’s silencing machinery. Deep-sequencing of small RNAs from plants infected with BYDV-PAV revealed that the virus is subjected to RNA silencing in the phloem tissues and there was no evidence of protection afforded by a possible decoy effect of the highly abundant subgenomic RNA3. However, analysis of VSR activity among the BYDV-PAV ORFs revealed systemic silencing suppression by the P4 movement protein, and a similar, but weaker, activity by P6. The closely related BYDV-PAS P4, but not the polerovirus potato leafroll virus P4, also displayed systemic VSR activity. Both luteovirus and the polerovirus P4 proteins also showed transient, weak local silencing suppression. This suggests that systemic silencing suppression is the principal mechanism by which the luteoviruses BYDV-PAV and BYDV-PAS minimize the effects of the plant’s anti-viral defense.

Amplification of the Phytophthora infestans RG57 loci Facilitates in planta T-RFLP Identification of Late Blight Genotypes

Late blight, caused by the oomycete Phytophthora infestans (Mont.) de Bary, is a devastating disease in potato and tomato and causes yield and quality losses worldwide. The disease first emerged in central America and has since spread in North America including the United States and Canada. Several new genotypes of P. infestans have recently emerged, including US-22, US-23 and US-24. Due to significant economic and environmental impacts, there has been an increasing interest in the rapid identification of P. infestans genotypes. In addition to providing details regarding the various phenotypic characteristics such as fungicide resistance, host preference, and pathogenicity associated with various P. infestans genotypes, information related to pathogen movement and potential recombination may also be determined from the genetic analyses. Restriction fragment length polymorphism (RFLP) analysis with the RG57 loci is one of the most reliable procedures used to genotype P. infestans. However, the RFLP procedure requires propagation and isolation of the pathogen and relatively large amounts of DNA. Isolation of the late blight pathogen is sometimes impossible due to the poor condition of the infected tissues or the presence of fungicide residues. In this study, we describe a procedure to identify P. infestans at the molecular level in planta using terminal restriction fragment length polymorphism (T-RFLP) of the RG57 loci. This T-RFLP assay is sufficiently sensitive to detect and differentiate P. infestans genotypes directly in planta without propagation and isolation of the pathogen, to facilitate the timely implementation of best management practices.ResumenEl tizón tardío, causado por el oomiceto Phytophthora infestans (Mont.) de Bary, es una enfermedad devastadora en papa y tomate, y causa pérdidas en rendimiento y calidad en todo el mundo. La enfermedad surgió primero en América Central y desde entonces se ha dispersado en Norteamérica, incluyendo los Estados Unidos y Canadá. Recientemente han emergido varios nuevos genotipos de P. infestans, incluyendo el US-22, US-23 y el US-24. Debido al impacto económico y ambiental significativos, ha habido un interés en aumento en la identificación rápida de los genotipos de P. infestans. Además de proporcionar detalles en relación a varias características fenotípicas, como la resistencia a fungicidas, preferencia de hospedante, y patogenicidad asociada a varios genotipos de P. infestans, la información relacionada al movimiento del patógeno y la recombinación potencial también pudiera determinarse de los análisis genéticos. El análisis del polimorfismo de longitud de fragmentos de restricción (RFLP) con el loci RG57 es uno de los procedimientos más confiables usados para el genotipo de P. infestans. No obstante, el procedimiento de RFLP requiere propagación y aislamiento del patógeno y relativamente grandes cantidades de ADN. El aislamiento del patógeno del tizón tardío algunas veces es imposible debido a la pobre condición de los tejidos infectados, o por la presencia de residuos de fungicidas. En este estudio, describimos un procedimiento para identificar P. infestans a nivel molecular in planta, usando polimorfismo terminal de longitud de fragmentos de restricción (T-RFLP) del loci RG57. Este ensayo de T-RFLP es lo suficientemente sensible para detectar y diferenciar genotipos de P. infestans directamente in planta sin la propagación y aislamiento del patógeno, para facilitar la implementación oportuna de las mejores prácticas de manejo.