A guide to the contained use of plant virus infectious clones.

Plant virus infectious clones are important tools with wide-ranging applications in different areas of biology and medicine. Their uses in plant pathology include the study of plant-virus interactions, and screening of germplasm as part of prebreeding programmes for virus resistance. They can also be modified to induce transient plant gene silencing (Virus Induced Gene Silencing - VIGS) and as expression vectors for plant or exogenous proteins, with applications in both plant pathology and more generally for the study of plant gene function. Plant viruses are also increasingly being investigated as expression vectors for in planta production of pharmaceutical products, known as molecular farming. However, plant virus infectious clones may pose a risk to the environment due to their ability to reconstitute fully functional, transmissible viruses. These risks arise from both their inherent pathogenicity and the effect of any introduced genetic modifications. Effective containment measures are therefore required. There has been no single comprehensive review of the biosafety considerations for the contained use of genetically modified plant viruses, despite their increasing importance across many biological fields. This review therefore explores the biosafety considerations for working with genetically modified plant viruses in contained environments, with focus on plant growth facilities. It includes regulatory frameworks, risk assessment, assignment of biosafety levels, facility features and working practices. The review is based on international guidance together with information provided by plant virus researchers.

Host to a Stranger: Arabidopsis and Fusarium Ear Blight.

Fusarium ear blight (FEB) is a devastating fungal disease of cereal crops. Outbreaks are sporadic and current control strategies are severely limited. This review highlights the use of Arabidopsis to study plant-FEB interactions. Use of this pathosystem has identified natural variation in Fusarium susceptibility in Arabidopsis, and native plant genes and signalling processes modulating the interaction. Recent breakthroughs include the identification of plant- and insect-derived small molecules which increase disease resistance, and the use of a host-induced gene silencing (HIGS) construct to silence an important Fusarium gene to prevent infection. Arabidopsis has also been used to study other fungi that cause cereal diseases. These findings offer the potential for translational research in cereals which could yield much-needed novel control strategies.

Mutations in the Arabidopsis homoserine kinase gene DMR1 confer enhanced resistance to Fusarium culmorum and F. graminearum.

BACKGROUND: Mutation of Arabidopsis DMR1, encoding homoserine kinase, leads to elevation in homoserine and foliar resistance to the biotrophic pathogens Hyaloperonospora arabidopsidis and Oidium neolycopersici through activation of an unidentified defence mechanism. This study investigates the effect of mutation of dmr1 on resistance to the ascomycete pathogens Fusarium graminearum and F. culmorum, which cause Fusarium Ear Blight (FEB) disease on small grain cereals. RESULTS: We initially found that the dmr1-2 mutant allele confers increased resistance to F. culmorum and F. graminearum silique infection, and decreased colonisation of rosette leaves. Meanwhile the dmr1-1 allele supports less rosette leaf colonisation but has wild type silique resistance. Three additional dmr1 alleles were subsequently examined for altered F. culmorum susceptibility and all showed increased silique resistance, while leaf colonisation was reduced in two (dmr1-3 and dmr1-4). Amino acid analysis of dmr1 siliques revealed homoserine accumulation, which is undetectable in wild type plants. Exogenous application of L-homoserine reduced bud infection in both dmr1 and wild type plants, whilst D-homoserine application did not. Delayed leaf senescence was also observed in dmr1 plants compared to wild type and correlated with reduced Fusarium leaf colonisation. CONCLUSIONS: These findings suggest that common Arabidopsis DMR1 mediated susceptibility mechanisms occur during infection by both obligate biotrophic oomycete and hemi-biotrophic fungal pathogens, not only in vegetative but also in reproductive plant tissues. This has the potential to aid the development of cereal crops with enhanced resistance to FEB.

Characterization of the sterol 14α-demethylases of Fusarium graminearum identifies a novel genus-specific CYP51 function.

CYP51 encodes the cytochrome P450 sterol 14α-demethylase, an enzyme essential for sterol biosynthesis and the target of azole fungicides. In Fusarium species, including pathogens of humans and plants, three CYP51 paralogues have been identified with one unique to the genus. Currently, the functions of these three genes and the rationale for their conservation within the genus Fusarium are unknown. Three Fusarium graminearum CYP51s (FgCYP51s) were heterologously expressed in Saccharomyces cerevisiae. Single and double FgCYP51 deletion mutants were generated and the functions of the FgCYP51s were characterized in vitro and in planta. FgCYP51A and FgCYP51B can complement yeast CYP51 function, whereas FgCYP51C cannot. FgCYP51A deletion increases the sensitivity of F. graminearum to the tested azoles. In ΔFgCYP51B and ΔFgCYP51BC mutants, ascospore formation is blocked, and eburicol and two additional 14-methylated sterols accumulate. FgCYP51C deletion reduces virulence on host wheat ears. FgCYP51B encodes the enzyme primarily responsible for sterol 14α-demethylation, and plays an essential role in ascospore formation. FgCYP51A encodes an additional sterol 14α-demethylase, induced on ergosterol depletion and responsible for the intrinsic variation in azole sensitivity. FgCYP51C does not encode a sterol 14α-demethylase, but is required for full virulence on host wheat ears. This is the first example of the functional diversification of a fungal CYP51.