In this doctoral study, we aimed to determine V. nonalfalfae candidate effectors, by analysing the available genomic and proteomic data, and the results of RNA-Seq experiments, and to confirm them with mutational analysis. From the annotated genetic models of V. nonalfafae (9269), we initially identified in silico secretome (944) enriched with enzymes involved in the degradation of cell walls, with proteases, lipases, cutinases and oxidoreductases, which corresponds to the hemibiotrophic life style of V. nonalfalfae. We further identified gene models expressed in planta (766), predicted candidate secretory effector proteins (263), and selected the best candidates based on the properties of already confirmed fungal effectors. We determined the expression of the most promising (44) candidates by RT-qPCR in resistant and susceptible varieties of hop after infection with V. nonalfalfae. Using the ATMT method, we prepared five deletion mutants of candidates with the highest expression, and artificially inoculated susceptible varieties of hop. Disease symptoms were assessed with a disease severity index and rAUDPC. The bioinformatic pipeline for determination of effectors proved to be relatively effective, since we determined previously verified V. nonalfalfae effectors VnaSSP4.2 and VnaUn.279. The deletion mutant ?Vna1.565 showed delayed disease symptoms and ?Vna8.691 showed increased virulence, while the remaining three deletion mutants did not show statistically significant differences compared to the wild type fungus, despite their high expression in planta. Their potential role in the pathogenesis of fungi is discussed.
D.09 Tutoring for postgraduate students
COBISS.SI-ID: 926327In the presented doctoral thesis, we characterized candidate effector proteins VnaChtBP and VnaSSP4.2, which were selected based on previous studies. Using confocal microscopy, a nucleo-cytosolic subcellular localization was determined for both effectors expressed in model plant Nicotiana benthamiana. Infiltration of recombinant proteins to various plants did not confirm the hypothesis of VnaChtBP or VnaSSP4.2 involvement in PTI or ETI responses. We were able to show that the recombinant protein VnaSSP4.2 binds to PIP, cadiolipin and sulfatide molecules, all known modulators of plant immunity. We determined that the effector VnaChtBP binds specifically to chitin and protects the fungus from degradation by plant chitinases.
D.09 Tutoring for postgraduate students
COBISS.SI-ID: 933239With the advent of high-throughput “-omics” technologies, increasing research focused on the fungus genome and its interaction with plant hosts and belowground microbiomes have surfaced. In addition, hundreds of pathogen effectors, secreted molecules that sabotage host machinery during the infection process, have been identified. Some of these effectors serve as valuable tools in effectoromics and are being exploited for accelerated and improved identification of immune receptors in modern resistance breeding. Pathogenomics greatly contributes to the in-depth understanding of the mechanisms underlying pathogenicity and virulence of the pathogen as well as defense and resistance of the host, thus paving the way for novel control strategies. Due to its recent emergence, the pathogenomics of Verticillium species is as young as it is challenging, and contributions in this field are being produced by scientists all around the world. Coordination actions, joint efforts, and open discussions would serve to better orientate investigations towards the shared objective of controlling this pathogen effectively.
C.03 Guest-associated editor
COBISS.SI-ID: 523338009The methods for agroinfiltration and effector localization were transferred to practical course of 3rd year studies of Biotechnology, subject Plant Biotechnology.
D.10 Educational activities
COBISS.SI-ID: 9344377From our laboratory, we transferred the method of agroinfiltration and detached leaf assay from N. benthamiana and Verticillium nonalfalfae to another pathosystem at the request of users from the Agricultural Institute. Thus, they were able to successfully select potato plants with the R8 resistance gene.
F.18 Transfer of new know-how to direct users (seminars, fora, conferences)
COBISS.SI-ID: 5818728