For their successful host colonization, plant pathogens employ various secreted proteins to suppress host immunity. Identification and characterization of pathogen-secreted proteins can contribute to an understanding of the pathogenicity mechanism and help in disease control. We used proteomics to search for proteins secreted to xylem by the vascular pathogen Verticillium nonalfalfae (formerly V. alboatrum) during colonization of hop plants. Three highly abundant fungal proteins were identified: two enzymes, α-Narabinofuranosidase and peroxidase, and one small secreted hypothetical protein. These are the first secreted proteins so far identified in xylem sap following infection with Verticillium spp. In planta expression of peroxidase and hypothetical protein genes increased with the progression of colonization, implicating their role in fungal virulence. Indeed, V. nonalfalfae deletion mutants of both genes exhibited attenuated virulence on hop plants, which returned to the level of the wild-type pathogenicity in the knockout complementation lines, supporting peroxidase and small secreted hypothetical protein as virulence factors required to promote V. nonalfalfae colonization of hop plants.
COBISS.SI-ID: 8365945
Hop (Humulus lupulus L.) plants are grown primarily for the brewing industry and have been used as a traditional medicinal herb for a long time. Severe hop stunt disease caused by the recently discovered Citrus bark cracking viroid (CBCVd) is one of the most devastating diseases among other viroid infections in hop. MicroRNAs (miRNAs) are a class of non-coding small RNAs that play important roles in gene expression regulation. To identify miRNAs in hop and their response to CBCVd-infection, two small RNA (sRNA) libraries were prepared from healthy and CBCVd-infected hop plants and were investigated by high throughput sequencing. A total of 67 conserved and 49 novel miRNAs were identified. Among them, 36 conserved and 37 novel miRNAs were found to be differentially recovered in response to CBCVd-infection. A total of 311 potential targets was predicted for conserved and novel miRNAs based on a sequence homology search using hop transcriptome data. The majority of predicted targets significantly belonged to transcriptional factors that may regulate hop leaf, root and cone growth and development. In addition, the identified miRNAs might also play an important roles in other cellular and metabolic processes, such as signal transduction, stress response and other physiological processes, including prenylflavonoid biosynthesis pathways. Quantitative real time PCR analysis of selected targets revealed their negative correlation with their corresponding CBCVd-responsive miRNAs. Based on the results, we concluded that CBCVd-responsive miRNAs modulate several hormone pathways and transcriptional factors that play important roles in the regulation of metabolism, growth and development. These results provide a framework for further analysis of regulatory roles of sRNAs in plant defense mechanism including other hop infecting viroids in particular.
COBISS.SI-ID: 8522361
Verticillium nonalfalfae is a fungal plant pathogen that causes wilt disease by colonizing the vascular tissues of host plants. The disease induced by hop isolates of V. nonalfalfae manifests in two different forms, ranging from mild symptoms to complete plant dieback, caused by mild and lethal pathotypes, respectively. Pathogenicity variations between the causal strains have been attributed to differences in genomic sequences and perhaps also to differences in their mitochondrial genomes. We used data from our recent Illumina NGS-based project of genome sequencing V. nonalfalfae to study the mitochondrial genomes of its different strains. The aim of the research was to prepare a V. nonalfalfae reference mitochondrial genome and to determine its phylogenetic placement in the fungal kingdom. The resulting 26,139 bp circular DNA molecule contains a full complement of the 14 "standard" fungal mitochondrial protein-coding genes of the electron transport chain and ATP synthase subunits, together with a small rRNA subunit, a large rRNA subunit, which contains ribosomal protein S3 encoded within a type IA-intron and 26 tRNAs. Phylogenetic analysis of this mitochondrial genome placed it in the Verticillium spp. lineage in the Glomerellales group, which is also supported by previous phylogenetic studies based on nuclear markers. The clustering with the closely related Verticillium dahliae mitochondrial genome showed a very conserved synteny and a high sequence similarity. Two distinguishing mitochondrial genome features were also found—a potential long non-coding RNA (orf414) contained only in the Verticillium spp. of the fungal kingdom, and a specific fragment length polymorphism observed only in V. dahliae and V. nubilum of all the Verticillium spp., thus showing potential as a species-specific biomarker.
COBISS.SI-ID: 8358265
In this review, several methods for viroid detection in various host plants are discussed, including their advantages and disadvantages. Even though relatively new molecular methods enable fast and sensitive detection of viroids, a combination of different methods gives the most reliable identification. Techniques based on nucleic acids may be the future for viroid detection but they still cannot replace biological indexing, which is usually essential in epidemiological and aetiological studies.
COBISS.SI-ID: 8472953
Drought is one of the major environmental devastating stressors that impair the growth and productivity of crop plants. Despite the relevance of drought stress, changes in physiology and resistance mechanisms are not completely understood for certain crops, including hop (Humulus lupulus L.). In this research the drought response of hop was studied using a conventional physiological approach (gas exchange techniques, fluorescence, relative water content measurements) and proteomic analysis (2D-DIGE). Plants of two cultivars (Aurora and Savinjski golding) were exposed to progressive drought in a pot experiment and analysed at different stress stages (mild, moderate and severe). Measurements of relative water content revealed a hydrostable water balance of hop. Photosynthesis was decreased due to stomatal and non-stomatal limitation to the same extent in both cultivars. Of 28 identified differentially abundant proteins, the majority were down regulated and included in photosynthetic (41%) and sugar metabolism (33%). Fifteen % of identified proteins were classified into the nitrogen metabolism, 4% were related to a ROS related pathway and 7% to other functions.
COBISS.SI-ID: 8388473