Francine H. Ishikawa

Heterokaryon Incompatibility Is Suppressed Following Conidial Anastomosis Tube Fusion in a Fungal Plant Pathogen

It has been hypothesized that horizontal gene/chromosome transfer and parasexual recombination following hyphal fusion between different strains may contribute to the emergence of wide genetic variability in plant pathogenic and other fungi. However, the significance of vegetative (heterokaryon) incompatibility responses, which commonly result in cell death, in preventing these processes is not known. In this study, we have assessed this issue following different types of hyphal fusion during colony initiation and in the mature colony. We used vegetatively compatible and incompatible strains of the common bean pathogen Colletotrichum lindemuthianum in which nuclei were labelled with either a green or red fluorescent protein in order to microscopically monitor the fates of nuclei and heterokaryotic cells following hyphal fusion. As opposed to fusion of hyphae in mature colonies that resulted in cell death within 3 h, fusions by conidial anastomosis tubes (CAT) between two incompatible strains during colony initiation did not induce the vegetative incompatibility response. Instead, fused conidia and germlings survived and formed heterokaryotic colonies that in turn produced uninucleate conidia that germinated to form colonies with phenotypic features different to those of either parental strain. Our results demonstrate that the vegetative incompatibility response is suppressed during colony initiation in C. lindemuthianum. Thus, CAT fusion may allow asexual fungi to increase their genetic diversity, and to acquire new pathogenic traits.

Live-cell imaging of conidial fusion in the bean pathogen, Colletotrichum lindemuthianum.

Fusion of conidia and conidial germlings by means of conidial anastomosis tubes (CATs) is a common phenomenon in filamentous fungi, including many plant pathogens. It has a number of different roles, and has been speculated to facilitate parasexual recombination and horizontal gene transfer between species. The bean pathogen Colletotrichum lindemuthianum naturally undergoes CAT fusion on the host surface and within asexual fruiting bodies in anthracnose lesions on its host. It has not been previously possible to analyze the whole process of CAT fusion in this or any other pathogen using live-cell imaging techniques. Here we report the development of a robust protocol for doing this with C. lindemuthianum in vitro. The percentage of conidial germination and CAT fusion was found to be dependent on culture age, media and the fungal strain used. Increased CAT fusion was correlated with reduced germ tube formation. We show time-lapse imaging of the whole process of CAT fusion in C. lindemuthianum for the first time and monitored nuclear migration through fused CATs using nuclei labelled with GFP. CAT fusion in this pathogen was found to exhibit significant differences to that in the model system Neurospora crassa. In contrast to N. crassa, CAT fusion in C. lindemuthianum is inhibited by nutrients (it only occurs in water) and the process takes considerably longer.

Genetic divergence among and within Colletotrichum lindemuthianum races assessed by RAPD

A divergencia genetica entre racas de Colletotrichum lindemuthianum foi obtida utilizando marcadores RAPD. Alem das diferentes racas deste fungo, foram incluidos neste estudo tres isolados da fase sexuada de Colletotrichum lindemuthianum (Glomerella cingulata f.sp. phaseoli). A informacao do padrao de bandas geradas utilizando 11 primers, permitiu a identificacao de 133 bandas polimorficas. As bandas polimorficas foram utilizadas para determinar a divergencia genetica entre e dentro de racas do patogeno. Os isolados analisados foram distribuidos em seis grupos com similaridade relativa de 0,75. O grupo VI, formado pelos tres isolados da fase sexual do Colletotrichum lindemuthianum, foi o mais divergente. As racas agrupadas previamente pelo uso de cultivares diferenciadoras nao apresentou qualquer relacao com a classificacao obtida pelos marcadores RAPD.

Genetic variability within isolates of Colletotrichum lindemuthianum belonging to race 65 from the state of Minas Gerais, Brazil

Colletotrichum lindemuthianum, the causal agent of anthracnose in the common bean (Phaseolus vulgaris), presents a wide genetic and pathogenic variability that gives rise to complications in the development of resistant bean cultivars. The aim of this study was to identify the variability within race 65 of C. lindemuthianum, the race most commonly encountered in Brazil, through randomly amplified polymorphic DNA (RAPD) and anastomosis analyses. Thirteen isolates of race 65, collected in different years and from various host cultivars located in diverse areas of the state of Minas Gerais, Brazil, were investigated. Twenty-four RAPD primers were employed and 83 polymorphic bands amplified. Genetic similarities were estimated from the Sorensen-Dice coefficient and ranged from 0.54 to 0.82. The dendrogram obtained by cluster analysis classified the isolates into 11 separate groups. For the purposes of the analysis of anastomosis, isolates were considered to be compatible when the fusion of hyphae from different isolates could be observed. The proportion of compatible reactions for each isolate was estimated and similarity estimates, based on the Russel & Rao coefficient, ranged from 0.28 to 0.85. Isolates were classified into 11 anastomosis groups, 10 of which were formed by only one isolate. Although isolates LV61, LV73 and LV58 were classified in the same anastomosis group, they were genetically distinct according to RAPD analysis. Results from both RAPD and anastomosis analyses revealed great variability within C. lindemuthianum race 65.

Factors affecting the production and regeneration of protoplasts from Colletotrichum lindemuthianum

O presente trabalho apresenta os fatores que afetam a producao e regeneracao de protoplastos de Colletotrichum lindemuthianum. O isolamento de protoplastos e muito relevante para diferentes aplicacoes, principalmente, em procedimentos que envolvem a manipulacao genetica. Estabilizadores osmoticos, enzimas liticas, tempo de incubacao e idade micelial foram testados com relacao ao efeito na liberacao de protoplastos. As condicoes otimizadas para producao de protoplastos foram incubacao de micelio jovem (48 h) em estabilizador osmotico NaCl 0.6 mol l-1, acrescido de 30 mg ml-1 da enzima Lysing Enzymes de Trichoderma harzianum incubado, durante 3 h. Nessas condicoes, a obtencao de protoplastos foi maior que 106 protoplatos ml-1 na mistura de digestao, numero suficientemente adequado para experimentos de transformacao em fungos. Sacarose nas concentracoes de 1.2 mol l-1 e 1 mol l-1 foram os estabilizadores mais apropriados para a regeneracao, apos 48 h, sendo as taxas de regeneracao de 16.35% e 14.54%, respectivamente. Este estudo produziu um metodo eficiente para producao e reversao de protoplastos a morfologia micelial tipica de Colletotrichum lindemuthianum utilizando o isolado LV115.

Colletotrichum lindemuthianum exhibits different patterns of nuclear division at different stages in its vegetative life cycle

Live-cell imaging with fluorescent protein labeling is providing major new insights into nuclear dynamics in filamentous fungi. With this approach we provide a detailed report of nuclear organization and behavior during mitosis in the bean pathogen Colletotrichum lindemuthianum. Nuclear division and nuclear migration were analyzed in ungerminated conidia, conidial germlings and the mature colony. Ungerminated conidia were uninucleate and completion of mitosis was found not to be essential for germ tube formation, conidial anastomosis tube (CAT) formation or fusion. Nuclei in fused conidial germlings exhibited asynchronous mitoses, and nuclear migration through fused CATs occurred after the nuclei had divided. Different patterns of nuclear division were found in vegetative hyphae of the mature colony. Synchronous, parasynchronous and asynchronous patterns of mitosis were observed in apical hyphal compartments at the colony border, while only synchronous and asynchronous mitoses occurred in subapical hyphal compartments. These findings have revealed unexpected diversity in the patterns of mitosis in different cells of C. lindemuthianum.