Search Results (4)

Microbotryum lychnidis-dioicae is an obligate fungal species colonizing the plant host, Silene latifolia. The fungus synthesizes and secretes effector proteins into the plant host during infection to manipulate the host for completion of the fungal lifecycle. The goal of this study was to continue functional characterization of such M. lychnidis-dioicae effectors. Here, we identified three putative effectors and their putative host-plant target proteins. MVLG_02245 is highly upregulated in M. lychnidis-dioicae during infection; yeast two-hybrid analysis suggests it targets a tubulin α-1 chain protein ortholog in the host, Silene latifolia. A potential plant protein interacting with MVLG_06175 was identified as CASP-like protein 2C1 (CASPL2C1), which facilitates the polymerization of the Casparian strip at the endodermal cells. Proteins interacting with MVLG_05122 were identified as CSN5a or 5b, involved in protein turnover. Fluorescently labelled MVLG_06175 and MVLG_05122 were expressed in the heterologous plant, Arabidopsis thaliana. MVLG_06175 formed clustered granules at the tips of trichomes on leaves and in root caps, while MVLG_05122 formed a band structure at the base of leaf trichomes. Plants expressing MVLG_05122 alone were more resistant to infection with Fusarium oxysporum. These results indicate that the fungus might affect the formation of the Casparian strip in the roots and the development of trichomes during infection as well as alter plant innate immunity. Full article

(1) Background: Silene latifolia is a dioecious plant, whose sex is determined by XY-type sex chromosomes. Microbotryum lychnidis-dioicae is a smut fungus that infects S. latifolia plants and causes masculinization in female flowers, as if Microbotryum were acting as a sex-determining gene. Recent large-scale sequencing efforts have promised to provide candidate genes that are involved in the sex determination machinery in plants. These candidate genes are to be analyzed for functional characterization. A virus vector can be a tool for functional gene analyses; (2) Methods: To develop a viral vector system in S. latifolia plants, we selected Apple latent spherical virus (ALSV) as an appropriate virus vector that has a wide host range; (3) Results: Following the optimization of the ALSV inoculation method, S. latifolia plants were infected with ALSV at high rates in the upper leaves. In situ hybridization analysis revealed that ALSV can migrate into the flower meristems in S. latifolia plants. Successful VIGS (virus-induced gene silencing) in S. latifolia plants was demonstrated with knockdown of the phytoene desaturase gene. Finally, the developed method was applied to floral organ genes to evaluate its usability in flowers; (4) Conclusion: The developed system enables functional gene analyses in S. latifolia plants, which can unveil gene functions and networks of S. latifolia plants, such as the mechanisms of sex determination and fungal-induced masculinization. Full article

(1) Background: Plant pathogenic fungi often display high levels of host specificity and biotrophic fungi; in particular, they must manipulate their hosts to avoid detection and to complete their obligate pathogenic lifecycles. One important strategy of such fungi is the secretion of small proteins that serve as effectors in this process. Microbotryum violaceum is a species complex whose members infect members of the Caryophyllaceae; M. lychnidis-dioicae, a parasite on Silene latifolia, is one of the best studied interactions. We are interested in identifying and characterizing effectors of the fungus and possible corresponding host targets; (2) Methods: In silico analysis of the M. lychnidis-dioicae genome and transcriptomes allowed us to predict a pool of small secreted proteins (SSPs) with the hallmarks of effectors, including a lack of conserved protein family (PFAM) domains and also localized regions of disorder. Putative SSPs were tested for secretion using a yeast secretion trap method. We then used yeast two-hybrid analyses for candidate-secreted effectors to probe a cDNA library from a range of growth conditions of the fungus, including infected plants; (3) Results: Roughly 50 SSPs were identified by in silico analysis. Of these, 4 were studied further and shown to be secreted, as well as examined for potential host interactors. One of the putative effectors, MVLG_01732, was found to interact with Arabidopsis thaliana calcium-dependent lipid binding protein (AtCLB) and with cellulose synthase interactive protein 1 orthologues; and (4) Conclusions: The identification of a pool of putative effectors provides a resource for functional characterization of fungal proteins that mediate the delicate interaction between pathogen and host. The candidate targets of effectors, e.g., AtCLB, involved in pollen germination suggest tantalizing insights that could drive future studies. Full article

Dioecious plants in the Caryophyllaceae family are susceptible to infection by members of the anthericolous smut fungi. In our studies of the Silene latifolia/Microbotryum lychnidis-dioicae pathosystem, we were interested in characterizing the plant-pathogen interaction at the molecular level before and during teliosporogenesis. This takes place during floral bud development, and we hoped to capture the interaction by Illumina Next-Gen RNA-Sequencing. Using previous literature that documented the stages of the floral buds for S. latifolia, we examined the floral buds from plants grown and infected under growth chamber conditions, using the disserting microscope to determine the stage of floral buds based on the morphology. We compiled the information and determined the size of floral buds that correspond to the desired stages of development for tissue collection, for the purpose of RNA-sequencing. This offers a practical approach for researchers who require a large number of floral buds/tissue categorized by stages of development, ascertaining whether infected/uninfected buds are at comparable stages of development and whether this also holds true for male vs. female buds. We also document our experience in infecting the plants and some of the unusual morphologies we observed after infection. Full article