Secretory proteins are delivered to the septin-organized penetration interface during root infection by Verticillium dahliae.

Successful infection of the host requires secretion of effector proteins to evade or suppress plant immunity. Secretion of effectors in root-infecting fungal pathogens, however, remains unexplored. We previously reported that Verticillium dahliae, a root-infecting phytopathogenic fungus, develops a penetration peg from a hyphopodium to infect cotton roots. In this study, we report that a septin ring, requiring VdSep5, partitions the hyphopodium and the invasive hypha and form the specialized fungus-host interface. The mutant strain, VdΔnoxb, in which NADPH oxidase B (VdNoxB) is deleted, impaired formation of the septin ring at the hyphal neck, indicating that NADPH oxidases regulate septin ring organization. Using GFP tagging and live cell imaging, we observed that several signal peptide containing secreted proteins showed ring signal accumulation/secretion at the penetration interface surrounding the hyphal neck. Targeted mutation for VdSep5 reduced the delivery rate of secretory proteins to the penetration interface. Blocking the secretory pathway by disrupting the vesicular trafficking factors, VdSec22 and VdSyn8, or the exocyst subunit, VdExo70, also arrested delivery of the secreted proteins inside the hyphopodium. Reduced virulence was observed when cotton roots were infected with VdΔsep5, VdΔsec22, VdΔsyn8 and VdΔexo70 mutants compared to infection with the isogenic wild-type V592. Taken together, our data demonstrate that the hyphal neck is an important site for protein secretion during plant root infection, and that the multiple secretory routes are involved in the secretion.

Verticillium dahliae transcription factors Som1 and Vta3 control microsclerotia formation and sequential steps of plant root penetration and colonisation to induce disease.

Verticillium dahliae nuclear transcription factors Som1 and Vta3 can rescue adhesion in a FLO8-deficient Saccharomyces cerevisiae strain. Som1 and Vta3 induce the expression of the yeast FLO1 and FLO11 genes encoding adhesins. Som1 and Vta3 are sequentially required for root penetration and colonisation of the plant host by V. dahliae. The SOM1 and VTA3 genes were deleted and their functions in fungus-induced plant pathogenesis were studied using genetic, cell biology, proteomic and plant pathogenicity experiments. Som1 supports fungal adhesion and root penetration and is required earlier than Vta3 in the colonisation of plant root surfaces and tomato plant infection. Som1 controls septa positioning and the size of vacuoles, and subsequently hyphal development including aerial hyphae formation and normal hyphal branching. Som1 and Vta3 control conidiation, microsclerotia formation, and antagonise in oxidative stress responses. The molecular function of Som1 is conserved between the plant pathogen V. dahliae and the opportunistic human pathogen Aspergillus fumigatus. Som1 controls genes for initial steps of plant root penetration, adhesion, oxidative stress response and VTA3 expression to allow subsequent root colonisation. Both Som1 and Vta3 regulate developmental genetic networks required for conidiation, microsclerotia formation and pathogenicity of V. dahliae.

Chitinolytic activity at low temperature of an Antarctic strain (A3) of Verticillium lecanii.

The chitinolytic activity of Verticillium cfr. lecanii A3, a strain isolated from continental Antarctica, was compared to those of two selected strains of Trichoderma harzianum. After 72 h of incubation at 25 degrees C in media containing chitin as the sole carbon source, all strains showed the same enzyme activity (ca. 230 mU/ml); at 15 degrees C, the levels of enzyme activity of the three strains were similar to those obtained at 25 degrees C. At 5 degrees C, in contrast, the activity of V. lecanii was ca. 4 times higher than those of both strains of T. harzianum (203 and 57 mU/ml, respectively; incubation time 144 h). The chitinase of V. lecanii, purified by preparative isoelectric focusing and ion-exchange chromatography, was shown to be a glycoprotein with apparent molecular weight of 45 kDa and isoelectric point of 4.9. The enzyme was active over a broad range of temperatures (5-60 degrees C): at 5 degrees C, its relative activity was still 50% of that recorded at 40 degrees C (optimal temperature). V. lecanii and its purified chitinase showed clear inhibitory effects on the growth of some test moulds such as Mucor plumbeus, Cladosporium cladosporioides, Aspergillus versicolor and Penicillium verrucosum: observations under the light and scanning electron microscopes revealed that growth inhibition was accompanied by mycelial damage and cell lysis.

Use of light and scanning electron microscopy to examine colonisation of barley rhizosphere by the nematophagous fungus Verticillium chlamydosporium.

Barley roots were readily colonised by the nematophagous fungus Verticillium chlamydosporium. Light microscopy (LM) but also low temperature scanning electron microscopy (LTSEM) revealed details of the colonisation process. Hyphae were found on the rhizoplane often with dictyochlamydospores. Hyphae of V. chlamydosporium penetrated epidermal cells, often by means of appressoria. A hyphal network was formed in epidermal and cortical cells. Likewise, hyphal coils were found within root cells next to transverse cell walls. Cortical cells were the limits of fungal colonisation, since no hyphae were seen in the vascular cylinder. Modifications of root cell contents (phenolic droplets and callose appositions) were common three weeks after inoculation with V. chlamydosporium. These features may indicate induction of plant defence reactions in late stages of root colonisation by the fungus. Both LTSEM and LM have proved extremely useful to describe root colonisation by the fungus. The results found may have implications in the mode action of nematophagous fungi against plant parasitic nematodes.

Morphology of Verticillium dahliae and V. tricorpus on semi-selective media used for the detection of V. dahliae in soil.

The morphology of two soil-borne Verticillium species, V. dahliae and V. tricorpus, was studied on two semi-selective agar media, in the absence and presence of soil. Morphology of the fungi differed considerably between the media, with respect to presence and shape of microsclerotia, dark hyphae (i.e. short melanised hyphae attached to the microsclerotia) and dark mycelium (i.e. melanised mycelium throughout the colony). On modified soil extract agar (MSEA), a pectate based agar, V. dahliae always had globose to elongate microsclerotia, without dark hyphae or dark mycelium, whereas V. tricorpus always had dark hyphae or dark mycelium, and microsclerotia, whenever present, were globose to irregular in shape. On ethanol agar (EA), V. dahliae had large microsclerotia and abundant dark hyphae, whereas V. tricorpus did not form microsclerotia, but always abundant dark mycelium. For the first time we observed the formation of dark hyphae by V. dahliae to a great extent. In the presence of soil, most characteristics were less pronounced, and V. dahliae microsclerotia were smaller, but V. tricorpus produced large microsclerotia, even when they were absent in pure culture. Morphological characteristics suitable for discrimination between the two species on MSEA plates in the presence of soil were selected and tested with fresh isolates from agricultural fields. The two fungi could be distinguished using qualitative characteristics and microsclerotial size. Molecular analysis and morphology on potato dextrose agar confirmed all identifications made on soil dilution plates.

Chemical components and their locations in the Verticillium fungicola cell wall.

The chemical structure of cell walls and fractions of Verticillium fungicola, a pathogen of Agaricus bisporus, as well as their corresponding ultrastructures were studied. There are at least three chemically distinct types of carbohydrate polymers: one yielding mannose with lower amounts of galactose and glucose (glucogalactomannan), another one composed mainly of glucose (glucan), and a third one containing only N-acetylglucosamine (chitin). Attempts were made to locate these materials in situ by comparing electron micrographs of shadowed and sectioned cell walls, and also by indirect immunofluorescence. It was shown that none of these polymers constituted a completely physically distinct layer, but there seem to be different solubility properties in the outer, inner, and intermediate layers. It was also shown that fibrillar material (chitin) embedded in cementing glucan constituted the residual inner fraction of the original wall material. Indirect immunofluorescence showed the location of a significant amount of glucogalactomannan on the surface of the walls in which rodlet structures were visualized by electron microscopy.