scholarly journals PM 7/78 (2) Verticillium nonalfalfae and V. dahliae

EPPO Bulletin ◽  
2020 ◽  
Vol 50 (3) ◽  
pp. 462-476
Keyword(s):  
Verticillium Nonalfalfae

scholarly journals Core RNA Interference Genes Involved in miRNA and Ta-siRNA Biogenesis in Hops and Their Expression Analysis after Challenging with Verticillium nonalfalfae

2021 ◽  
Vol 22 (8) ◽  
pp. 4224
Author(s):  
Urban Kunej ◽  
Jernej Jakše ◽  
Sebastjan Radišek ◽  
Nataša Štajner
Keyword(s):  
Rna Interference ◽  
Expression Analysis ◽  
Cannabis Sativa ◽  
Defense Responses ◽  
Resistant Cultivar ◽  
Post Inoculation ◽  
Biotic Stresses ◽  
Argonaute 2 ◽  
Expression Of Genes ◽  
Verticillium Nonalfalfae

RNA interference is an evolutionary conserved mechanism by which organisms regulate the expression of genes in a sequence-specific manner to modulate defense responses against various abiotic or biotic stresses. Hops are grown for their use in brewing and, in recent years, for the pharmaceutical industry. Hop production is threatened by many phytopathogens, of which Verticillium, the causal agent of Verticillium wilt, is a major contributor to yield losses. In the present study, we performed identification, characterization, phylogenetic, and expression analyses of three Argonaute, two Dicer-like, and two RNA-dependent RNA polymerase genes in the susceptible hop cultivar Celeia and the resistant cultivar Wye Target after infection with Verticillium nonalfalfae. Phylogeny results showed clustering of hop RNAi proteins with their orthologues from the closely related species Cannabis sativa, Morus notabilis and Ziziphus jujuba which form a common cluster with species of the Rosaceae family. Expression analysis revealed downregulation of argonaute 2 in both cultivars on the third day post-inoculation, which may result in reduced AGO2-siRNA-mediated posttranscriptional gene silencing. Both cultivars may also repress ta-siRNA biogenesis at different dpi, as we observed downregulation of argonaute 7 in the susceptible cultivar on day 1 and downregulation of RDR6 in the resistant cultivar on day 3 after inoculation.

Studying RNAi in pathogenic hop fungi Verticillium nonalfalfae

2021 ◽  
pp. 37-46
Author(s):  
T. Jeseničnik ◽  
N. Štajner ◽  
S. Radišek ◽  
A. Kumar-Mishra ◽  
J. Jakše
Keyword(s):  
Verticillium Nonalfalfae

scholarly journals Comprehensive analysis of Verticillium nonalfalfae in silico secretome uncovers putative effector proteins expressed during hop invasion

2017 ◽  
Author(s):  
Kristina Marton ◽  
Marko Flajšman ◽  
Sebastjan Radišek ◽  
Katarina Košmelj ◽  
Jernej Jakše ◽  
...  
Keyword(s):  
Verticillium Wilt ◽  
Sequence Similarity ◽  
Vascular Plant ◽  
Xylem Sap ◽  
Fungal Growth ◽  
Effector Proteins ◽  
Specific Gene ◽  
In Planta ◽  
Verticillium Nonalfalfae ◽  
Spatio Temporal

AbstractBackgroundThe vascular plant pathogen Verticillium nonalfalfae causes Verticillium wilt in several important crops. VnaSSP4.2 was recently discovered as a V. nonalfalfae virulence effector protein in the xylem sap of infected hop. Here, we expanded our search for candidate secreted effector proteins (CSEPs) in the V. nonalfalfae predicted secretome using a bioinformatic pipeline built on V. nonalfalfae genome data, RNA-Seq and proteomic studies of the interaction with hop.ResultsThe secretome, rich in carbohydrate active enzymes, proteases, redox proteins and proteins involved in secondary metabolism, cellular processing and signaling, includes 263 CSEPs. Several homologs of known fungal effectors (LysM, NLPs, Hce2, Cerato-platanins, Cyanovirin-N lectins, hydrophobins and CFEM domain containing proteins) and avirulence determinants in the PHI database (Avr-Pita1 and MgSM1) were found. The majority of CSEPs were non-annotated and were narrowed down to 44 top priority candidates based on their likelihood of being effectors. These were examined by spatio-temporal gene expression profiling of infected hop. Among the highest in planta expressed CSEPs, five deletion mutants were tested in pathogenicity assays. A deletion mutant of VnaUn.279, a lethal pathotype specific gene with sequence similarity to SAM-dependent methyltransferase (LaeA), had lower infectivity and showed highly reduced virulence, but no changes in morphology, fungal growth or conidiation were observed.ConclusionsSeveral putative secreted effector proteins that probably contribute to V. nonalfalfae colonization of hop were identified in this study. Among them, LaeA gene homolog was found to act as a potential novel virulence effector of V. nonalfalfae. The combined results will serve for future characterization of V. nonalfalfae effectors, which will advance our understanding of Verticillium wilt disease.

scholarly journals First Report of Verticillium Wilt Caused by Verticillium nonalfalfae on Tree-of-Heaven (Ailanthus altissima) in Ohio

Plant Disease ◽  
2013 ◽  
Vol 97 (7) ◽  
pp. 999-999 ◽  
Author(s):  
J. Rebbeck ◽  
M. A. Malone ◽  
D. P. G. Short ◽  
M. T. Kasson ◽  
E. S. O'Neal ◽  
...  
Keyword(s):  
Verticillium Wilt ◽  
Elongation Factor ◽  
The United States ◽  
Selective Medium ◽  
Ailanthus Altissima ◽  
Tryptophan Synthase ◽  
Protein Coding ◽  
Pike County ◽  
Tree Of Heaven ◽  
Verticillium Nonalfalfae

Verticillium wilt of the highly invasive tree-of-heaven [Ailanthus altissima (Mill.) Swingle], caused by Verticillium nonalfalfae Interbitzin et al. (1), formerly classified as V. albo-atrum Reinke and Berthold, has been reported in the United States from two states: Pennsylvania (2) and Virginia (3). Infected A. altissima in both states exhibited similar symptoms of wilt, premature defoliation, terminal dieback, yellow vascular discoloration, and mortality. In June 2012, the second author observed dead and dying A. altissima trees in southern Ohio (Pike County) that exhibited symptoms similar to those on diseased A. altissima trees in Pennsylvania and Virginia. Samples were collected from stems of three symptomatic A. altissima trees and sent to Penn State for morphological and molecular identification. Immediately upon arrival, samples were surface-disinfected and plated onto plum extract agar (PEA), a semi-selective medium for Verticillium spp., amended with neomycin and streptomycin (2). The samples yielded six isolates, two from each of the three symptomatic trees, all of which were putatively identified as V. nonalfalfae based on the presence of verticillate conidiophores and formation of melanized hyphae. DNA was extracted from three isolates and molecular analyses performed using known primers (1) coding for elongation factor 1-alpha (EF), glyceraldehyde-3-phosphate dehydrogenase (GPD), and tryptophan synthase (TS). A BLAST search generated sequences that revealed 100% similarity to V. nonalfalfae for all three protein coding genes among the three Ohio isolates and reference sequences from Ailanthus, including isolates VnAaPA140 (GenBank Accession Nos. KC307764, KC307766, and KC307768) and VnAaVA2 (KC307758, KC307759, and KC307760), as well as isolate PD592 from potato (JN188227, JN188163, and JN188035), thereby confirming taxonomic placement of the Ohio Ailanthus isolates among those recovered from Ailanthus in Pennsylvania and Virginia. Aligned sequences from one representative isolate, VnAaOH1, were deposited into GenBank as accessions KC307761 (EF), KC307762 (GPD), and KC307763 (TS). In August 2012, the pathogenicity of all six isolates was confirmed by root-dipping 10 healthy 3-week-old A. altissima seedlings (seeds collected in University Park, PA) into conidial suspensions of 1 × 107 cfu/ml, wherein all inoculated seedlings wilted and died within 4 and 9 weeks, respectively. V. nonalfalfae was reisolated from all inoculated seedlings; control seedlings inoculated with distilled water remained asymptomatic. Ohio is the third state from which V. nonalfalfae has been reported to be pathogenic on A. altissima. If V. nonalfalfae proves to be widespread, it may represent a natural biocontrol for the invasive A. altissima. Also, since USDA APHIS evaluates and regulates new potential biocontrol agents on a state-by-state basis, it is important to document each state in which V. nonalfalfae is killing A. altissima, so that in-state inoculum can be used for biocontrol efforts, simplifying the regulatory process. References: (1) P. Inderbitzin et al. 2011 PLoS ONE, 6, e28341, 2011. (2) M. J. Schall and D. D. Davis. Plant Dis. 93:747, 2009. (3) A. L. Snyder et al. Plant Dis. 96:837, 2013.

scholarly journals Discovery of microRNA-like Small RNAs in Pathogenic Plant Fungus Verticillium nonalfalfae Using High-Throughput Sequencing and qPCR and RLM-RACE Validation

2022 ◽  
Vol 23 (2) ◽  
pp. 900
Author(s):  
Taja Jeseničnik ◽  
Nataša Štajner ◽  
Sebastjan Radišek ◽  
Ajay Kumar Mishra ◽  
Katarina Košmelj ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Expression Patterns ◽  
Pathogenic Fungi ◽  
Absolute Quantification ◽  
Stem Loop ◽  
Humulus Lupulus L ◽  
Verticillium Nonalfalfae ◽  
Highly Virulent ◽  
Plant Fungal Pathogen

Verticillium nonalfalfae (V. nonalfalfae) is one of the most problematic hop (Humulus lupulus L.) pathogens, as the highly virulent fungal pathotypes cause severe annual yield losses due to infections of entire hop fields. In recent years, the RNA interference (RNAi) mechanism has become one of the main areas of focus in plant—fungal pathogen interaction studies and has been implicated as one of the major contributors to fungal pathogenicity. MicroRNA-like RNAs (milRNAs) have been identified in several important plant pathogenic fungi; however, to date, no milRNA has been reported in the V. nonalfalfae species. In the present study, using a high-throughput sequencing approach and extensive bioinformatics analysis, a total of 156 milRNA precursors were identified in the annotated V. nonalfalfae genome, and 27 of these milRNA precursors were selected as true milRNA candidates, with appropriate microRNA hairpin secondary structures. The stem-loop RT-qPCR assay was used for milRNA validation; a total of nine V. nonalfalfae milRNAs were detected, and their expression was confirmed. The milRNA expression patterns, determined by the absolute quantification approach, imply that milRNAs play an important role in the pathogenicity of highly virulent V. nonalfalfae pathotypes. Computational analysis predicted milRNA targets in the V. nonalfalfae genome and in the host hop transcriptome, and the activity of milRNA-mediated RNAi target cleavage was subsequently confirmed for two selected endogenous fungal target gene models using the 5′ RLM-RACE approach.

scholarly journals Changes in the Phenolic Compounds of Hop (Humulus lupulus L.) Induced by Infection with Verticillium nonalfalfae, the Causal Agent of Hop Verticillium Wilt

Plants ◽  
2020 ◽  
Vol 9 (7) ◽  
pp. 841 ◽  
Author(s):  
Urban Kunej ◽  
Maja Mikulič-Petkovšek ◽  
Sebastjan Radišek ◽  
Nataša Štajner
Keyword(s):  
Phenolic Compounds ◽  
Verticillium Wilt ◽  
Quantitative Polymerase Chain Reaction ◽  
Humulus Lupulus ◽  
Stress Factors ◽  
Resistant Cultivar ◽  
Susceptible Cultivar ◽  
Humulus Lupulus L ◽  
Wide Range ◽  
Verticillium Nonalfalfae

Phenolic compounds are involved in plant responses to various biotic and abiotic stress factors, with many studies suggesting their role in defense mechanisms against fungal pathogens. Soilborne vascular pathogen Verticillium nonalfalfae causes severe wilting and consequent dieback in a wide range of economically important crops, including hops (Humulus lupulus L.). In this study, we investigated the differential accumulation of phenolics in the susceptible “Celeia” and resistant “Wye Target” hop cultivars during the pathogenesis of Verticillium wilt. Quantitative polymerase chain reaction showed that colonization in the roots of both cultivars was intensive, but decreased continuously throughout the experiment in the resistant cultivar, while the relative fungal amount continuously increased in the stems of the susceptible cultivar. In response to colonization in the roots of the resistant cultivar, a significant increase in total flavanols was detected at three days postinoculation (dpi), suggesting a possible role in preventing fungus spread into the stems. The accumulation of phenolic compounds was less pronounced in the stems of the resistant cultivar since, compared to the latter, significant increases in flavonols at 3 and 15 dpi and hydroxycinnamic acids at 6 dpi were observed in the stems of the susceptible cultivar.

The invisibility cloak: Chitin binding protein of Verticillium nonalfalfae disguises fungus from plant chitinases

2018 ◽  
Author(s):  
Helena Volk ◽  
Kristina Marton ◽  
Marko Flajšman ◽  
Sebastjan Radišek ◽  
Ingo Hein ◽  
...  
Keyword(s):  
Fungal Infections ◽  
Binding Protein ◽  
Homology Modelling ◽  
Binding Motif ◽  
Carbohydrate Binding ◽  
Fungal Cell ◽  
Verticillium Nonalfalfae ◽  
Chitin Binding Protein ◽  
Plant Chitinases

AbstractDuring fungal infections, plant cells secrete chitinases that digest chitin in the fungal cell walls. The recognition of released chitin oligomers via lysin motif (LysM)-containing immune receptors results in the activation of defence signalling pathways. We report here that Verticillium nonalfalfae, a hemibiotrophic xylem-invading fungus, prevents this recognition process by secreting a CBM18 (carbohydrate binding motif 18)-chitin binding protein, VnaChtBP, which is transcriptionally activated specifically during the parasitic life stages. VnaChtBP is encoded by the Vna8.213 gene which is highly conserved within the species, suggesting high evolutionary stability and importance for the fungal lifestyle. In a pathogenicity assay, however, Vna8.213 knockout mutants exhibit wilting symptoms similar to the wild type fungus, suggesting that Vna8.213 activity is functionally redundant during fungal infection of hop. In binding assay, recombinant VnaChtBP binds chitin and chitin oligomers in vitro with submicromolar affinity and protects fungal hyphae from degradation by plant chitinases. Using a yeast-two-hybrid assay, homology modelling and molecular docking, we demonstrated that VnaChtBP forms dimers in the absence of ligands and that this interaction is stabilized by the binding of chitin hexamers with a similar preference in the two binding sites. Our data suggest that, in addition to chitin binding LysM (CBM50) and Avr4 (CBM14) fungal effectors, structurally unrelated CBM18 effectors have convergently evolved to prevent hydrolysis of the fungal cell wall against plant chitinases.

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