Molecular dissection of perithecial mating line development in Colletotrichum fructicola, a species with a nontypical mating system featured by plus-to-minus switch and plus-minus mediated sexual enhancement

The genetic regulation of Colletotrichum (Glomerella) sexual reproduction does not strictly adhere to the Ascomycota paradigm and remains poorly understood. Morphologically different but sexually compatible strain types termed ‘plus’ and ‘minus’ have been recognized but the biological and molecular distinctions between these strain types remain elusive. In this study, we characterized the sexual behaviors of a pair of plus and minus strains of C. fructicola with the aid of live cell nuclear-localized fluorescent protein labeling, gene expression and gene mutation analyses. We confirmed a genetically stable plus-to-minus switching phenomenon and demonstrated the presence of both cross-fertilized and self-fertilized perithecia within the mating line (perithecia cluster at the line of colony contact) between plus and minus strains. We demonstrated that pheromone signaling genes (a-factor-like and α-factor-like pheromones and their corresponding GPCR receptors) were differently expressed between vegetative hyphae of the two strains. Moreover, deletion of pmk1 (a FUS/KSS1 MAPK) in the minus strain severely limited mating line formation whereas deletion of a GPCR (FGSG_05239 homolog) and two histone modification factors (hos2, snt2) in the minus strain did not affect mating line development, but altered the ratio between cross-fertilization and self-fertilization within the mating line. We propose a model that mating line formation in C. fructicola involves enhanced protoperithecium differentiation and enhanced perithecium maturation of the minus strain mediated by both cross-fertilization and diffusive effectors. This study provides insights into mechanisms underlying the mysterious phenomenon of plus-minus mediated sexual enhancement being unique to Colletotrichum fungi. IMPORTANCE Plus-minus regulation of Colletotrichum sexual differentiation was reported in the early 1900s. Both plus and minus strains produce fertile perithecia in a homothallic but inefficient manner. However, when the two strain types encounter each other, efficient differentiation of fertile perithecia is triggered. The plus strain, by itself, can also generate minus ascospore progeny at high frequency. This nontypical mating system facilitates sexual reproduction and is Colletotrichum-specific, the underlying molecular mechanisms, however, remain elusive. The current study revisits this longstanding mystery using C. fructicola as an experimental system. The presence of both cross-fertilized and self-fertilized perithecia within the mating line were directly evidenced by live cell imaging with fluorescent markers. Based on further gene expression and gene mutation analysis, a model explaining mating line development (plus-minus mediated sexual enhancement) is proposed. Data reported here has the potential to better understand Colletotrichum mating and filamentous ascomycete sexual regulation.

Plasmids associated with a phagelike particle and with a satellite inclusion in Bacillus thuringiensis ssp. israelensis

Variants of Bacillus thuringiensis ssp. israelensis were isolated using a 42 °C plasmid curing method. Those that were missing the large toxic crystalline inclusion still possessed in their sporulated cells a small inclusion similar to the satellite inclusion of the wild type. These crystal-minus but satellite-positive variants, like the wild type, produced sporulation-dependent phagelike particles; variants lacking both the crystal and the satellite did not. These latter strains lacked both a 75 and a 68 megadalton plasmid present in the wild type. However, a crystal-positive, satellite-minus strain possessing the 75 but lacking the 68 megadalton plasmid produced the phagelike particles. Examination of recipient strains that were initially devoid of crystal, satellite, and plasmids revealed the satellite and phagelike producing characteristics could have been cotransformed with the 68 megadalton plasmid. Likewise the toxic crystal and phagelike particle producing characters could have been cotransformed into a recipient which had acquired the 75 megadalton plasmids already known to be associated with the synthesis of the crystal inclusion. Thus the gene coding for the satellite inclusion appears to reside in the 68 megadalton plasmid, while that coding for the phagelike particle can reside in either the 68 or 75 megadalton plasmid.

A STUDY OF THE TRANSMISSION AND STRUCTURE OF DOUBLE STRANDED RNAs ASSOCIATED WITH THE KILLER PHENOMENON IN SACCHAROMYCES CEREVISIAE

ABSTRACT Killer strains contain two double stranded RNAs, L and M. The M dsRNA appears to be necessary for production of a toxin and for resistance to that toxin. Mutant strains have been found that are defective in their ability to kill and in their resistance to toxin. These sensitive, non-killer strains have altered dsRNA composition. One class has no M dsRNA. Another class of sensitive, non-killers called suppressives has no M dsRNA but instead has smaller dsRNAs called S. In diploids resulting from a cross of a wild-type killer by a suppressive the transmission of the M dsRNA is suppressed by the S dsRNA. When a suppressive is crossed by a strain with no M dsRNA, the diploids and all four meiotic spores have the S dsRNA characteristic of the parental suppressive strain. Suppressive strains do not suppress each other. Intercrosses between two different suppressives yields diploids with both parental S dsRNAs. These two S dsRNAs are transmitted to all 4 meiotic progeny. Another class of mutants has been found which is defective for one of the traits but retains the other. One type, temperature-sensitive killers, has a normal dsRNA composition but is unable to kill at 30°. The other type, immunity-minus, has a complex dsRNA pattern. The immunity-minus strain is extremely unstable during mitotic growth and segregates several different types of non-killers. Analysis of the dsRNAs from wild type and the mutants by electron microscopy shows that the L, M, and S dsRNAs are linear. All strains regardless of killer phenotype appear to have the same size L dsRNA.

INFLUENCE OF INSECT LURES ON HYPHAL GROWTH AND SPORULATION OF CHOANEPHORA TRISPORA

The influence of 10 insect lures on hyphal growth and asexual and sexual sporulation of the heterothallic fungus Choanephora trispora was determined. The least toxic lures were 1,2-hexadecanediol, 1,2-epoxy-hexadecane, and Gyplure, while Trimedlure, Medlure, and methyl eugenol were the most inhibitory to hyphal growth. In general, if an insect lure permitted hyphal growth, formation of some sporangia was observed. Under normal conditions the plus mating type formed more sporangia than did the minus strain. Sporangia formation of both mating types was stimulated by 1,2-hexadecanediol. Methyl eugenol (0.06%) was observed to disrupt and inhibit the positive chemotactic response of the hyphae for the opposite mating types which is observed prior to formation of zygospores. In addition, two other eugenol derivatives, 0.03% eugenol acetate and 0.003% isoeugenol formate, were found to inhibit inception of zygospores. Levels of eugenol which did not influence hyphal growth also did not inhibit carotene synthesis, which is interrelated with the sexual processes of this fungus. Isoeugenol formate partially inhibited sporangia formation in both mating types.