A Cytosine Methyltransferase Homologue Is Essential for Sexual Development in Aspergillus nidulans

Background: The genome defense processes RIP (repeat-induced point mutation) in the filamentous fungus Neurospora crassa, and MIP (methylation induced premeiotically) in the fungus Ascobolus immersus depend on proteins with DNA methyltransferase (DMT) domains. Nevertheless, these proteins, RID and Masc1, respectively, have not been demonstrated to have DMT activity. We discovered a close homologue in Aspergillus nidulans, a fungus thought to have no methylation and no genome defense system comparable to RIP or MIP. Principal Findings: We report the cloning and characterization of the DNA methyltransferase homologue A (dmtA) gene from Aspergillus nidulans. We found that the dmtA locus encodes both a sense (dmtA) and an anti-sense transcript (tmdA). Both transcripts are expressed in vegetative, conidial and sexual tissues. We determined that dmtA, but not tmdA, is required for early sexual development and formation of viable ascospores. We also tested if DNA methylation accumulated in any of the dmtA/tmdA mutants we constructed, and found that in both asexual and sexual tissues, these mutants, just like wild-type strains, appear devoid of DNA methylation. Conclusions/Significance: Our results demonstrate that a DMT homologue closely related to proteins implicated in RIP and MIP has an essential developmental function in a fungus that appears to lack both DNA methylation and RIP or MIP. It remains formally possible that DmtA is a bona fide DMT, responsible for trace, undetected DNA methylation that i

Liquid-Liquid Interfacial Imaging Using Atomic Force Microscopy

Nanoscale imaging of a liquid/liquid interface by atomic force microscopy (AFM) is achieved through the self-assembly of interfacial layers of either polymers, lipids, or nanoparticles. Stabilization of the interface through spreading of a thin film of polystyrene-b-poly(methyl-methacrylate) block copolymer or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine lipids or adsorption of monolayers of silica nanoparticles allows reproducible imaging of these soft materials at an oil/water interface with nanoscale resolution on a timescale of 10 s. Amplitude modulation AFM is employed and requires that the subphase of water is below a critical depth of 100 µm to prevent excitation of gravity waves at the interface. The amplitude of these vertical oscillations is of the order of 1 nm but increases with the water layer depth. Below this critical water layer depth, force measurements show a linear compliance of the water/heptane and water/octane interfaces to be close to 10−2 N m−1. This study discusses in detail the experimental setup, sample preparation procedures, and AFM parameters necessary to achieve nanoscale resolution at the extremely soft and dynamic liquid interface. This expands the application of AFM to structural and dynamic nanoscale measurements for soft matter, biological, and nanomaterials away from solid supports

MOLECULAR ANALYSIS OF PHYTOPHTHORA DIVERSITY IN ORNAMENTAL NURSERIES.

A molecular approach based on the use of genus-specific nested-PCR primers (Scibetta et al., 2012; J. Microbiol. Meth. 88: 356-368) was utilized to detect Phytophthora species in soil and root samples of potted ornamentals, collected across Apulia and Calabria, Southern Italy. Analyzed samples comprised many plant species with different levels of symptoms of decline on the canopy and root rots. Extraction protocols were optimized to obtain DNA samples of appropriate quality from soil and roots. The analysis of sequences after cloning of nested-PCR amplicons enabled the identification of different Phytophthora species including P. nicotianae, P. cinnamomi, P. cryptogea, P. palmivora and P. niederhauserii. Interestingly, a higher level of intraspecific variability was detected within each Phytophthora species if compared with results of previous reports obtained using the same method in natural ecosystems. Although, the existence of PCR artifacts due to Taq DNA polymerase errors cannot be completely excluded, the detection of the same genotypes (commonly differentiated by one or few polymorphic nucleotides) in different soil and/or root samples, confirmed the reliability of results. As a consequence the higher intraspecific variability detected in ornamental nurseries seem to be the results of a more intensive sexual recombination favored by the concurrent cultivation of many plant species which increases the meeting of different genetically distant isolates of the pathogens

Development of quantitative PCR detection methods for phytopathogenic fungi and oomycetes

In recent years quantitative PCR (qPCR) detection methods have been widely utilised to detect phytopathogenic fungi and oomycetes and have greatly contributed to the advancement of knowledge in plant pathology. However, major drawbacks and common errors, most typical of earlier reports, still affect many methods currently available in the literature. Errors can be made throughout the entire process for the development of qPCR methods, at the level of selection of appropriate DNA extraction and purification protocols, identification of suitable target regions, choice of the chemistry, design and validation of specific primers and probes, analysis of sensitivity, choice of an absolute and/or relative quantification approach and analysis of the risk of detecting target DNA from dead sources. In the present review the above mentioned steps are analysed, highlighting their critical aspects and providing a practical guide for the users