Asztéria Almási

Significance of hibernated Thrips tabaci Lindeman (Thysan., Thripidae) adults in the epidemic of tomato spotted wilt virus

Abstract:  Tomato spotted wilt tospovirus (TSWV) was transmitted to indicator plants by Thrips tabaci, and was detected even in single specimens collected from different plants from September until May in Hungary. Consequently, the specimens of the hibernating generation are able to harbour TSWV from autumn until spring, for 6 months. They could infect the young seedling in the forced beds, or in the field in May, and thereby they have a significant role in the TSWV epidemic in spring under Hungarian climatic conditions.

Phosphorylation regulates the subcellular localization of Cucumber Mosaic Virus 2b protein

The 2b protein of Cucumber mosaic virus has a role in nearly all steps of the viral cycle including cell-to-cell movement, symptom induction and suppression of antiviral RNA silencing. Previous studies demonstrated the presence of 2b protein in the nucleus and in cytoplasm as well. Phosphorylation site of 2b protein is conserved in all CMV isolates, including proposed constitute motifs for casein kinase II and cyclin-dependent kinase 2. To discern the impact of 2b protein phosphorylation, we created eight different mutants to mimic the non-phosporylated (serine to alanine) as well as the phosphorylated state (serine to aspartic acid) of the protein. We compared these mutants to the wild-type (Rs-CMV) virus in terms of symptom induction, gene silencing suppressor activity as well as in cellular localization. Here, in this study we confirmed the phosphorylation of 2b protein in vivo, both in infected N. benthamiana and in infiltrated patches. Mutants containing aspartic acid in the phosphorylation site accumulated only in the cytoplasm indicating that phosphorylated 2b protein could not enter the nucleus. We identified a conserved dual phosphorylation switch in CMV 2b protein, which equilibrates the shuttling of the 2b protein between the nucleus and the cytoplasm, and regulates the suppressor activity of the 2b protein.

Phylogenetic analysis of Tomato spotted wilt virus (TSWV) NSs protein demonstrates the isolated emergence of resistance-breaking strains in pepper

Resurgence of Tomato spotted wilt virus (TSWV) worldwide as well as in Hungary causing heavy economic losses directed the attention to the factors contributing to the outbreak of this serious epidemics. The introgression of Tsw resistance gene into various pepper cultivars seemed to solve TSWV control, but widely used resistant pepper cultivars bearing the same, unique resistance locus evoked the rapid emergence of resistance-breaking (RB) TSWV strains. In Hungary, the sporadic appearance of RB strains in pepper-producing region was first observed in 2010–2011, but in 2012 it was detected frequently. Previously, the non-structural protein (NSs) encoded by small RNA (S RNA) of TSWV was verified as the avirulence factor for Tsw resistance, therefore we analyzed the S RNA of the Hungarian RB and wild type (WT) isolates and compared to previously analyzed TSWV strains with RB properties from different geographical origins. Phylogenetic analysis demonstrated that the different RB strains had the closest relationship with the local WT isolates and there is no conserved mutation present in all the NSs genes of RB isolates from different geographical origins. According to these results, we concluded that the RB isolates evolved separately in geographic point of view, and also according to the RB mechanism.

S -methylmethionine contributes to enhanced defense against Maize dwarf mosaic virus infection in maize

The beneficial and protective effects of S-methylmethionine (SMM) were investigated in Maize dwarf mosaic virus (MDMV) infected maize (Zea mays L.). Response reactions and alterations in the physiological state of the plants were monitored by following changes in the values of chlorophyll a fluorescence and chlorophyll content, and differences in the expression patterns of the stress-related genes S-adenosylmethionine synthase (SAMS) and the 14-3-3-like protein gene G-box factor 14-6 (GF14-6). Infection was validated using the ELISA technique. A pronounced decrease in the red to far-red chlorophyll a fluorescence ratio, indicative of chlorophyll content, was observed in infected plants, which was tempered by SMM pretreatment. A noticeable decrease in the photochemical quenching of photosystem II and the thermal dissipation of the antennae was observed, together with a notable increase in other non-photochemical energy dissipation parameters in response to MDMV infection. SMM treatment enhanced blue fluorescence in both uninfected and MDMV-infected plants (probably due to the production of protective phenolic compounds), while infection characteristically increased green fluorescence emission. SMM treatment was found to elevate the rate of gene expressions of SAMS and GF14-6. The results suggest that SMM pretreatment enhances the stress response reactions that protect maize plants against MDMV infection.

A single point mutation in Tomato spotted wilt virus NSs protein is sufficient to overcome Tsw-gene-mediated resistance in pepper

The nonstructural protein (NSs) of Tomato spotted wilt virus (TSWV) was previously identified as an avirulence determinant for Tsw-based resistance on pepper. The NSs of wild-type (WT) and resistance-breaking (RB) TSWV strains isolated in Hungary had only two amino acid substitutions (104, 461). We have analysed the ability of the NSs and their point mutant variants to trigger Tsw-mediated hypersensitive responses and RNA silencing suppressor (RSS) activity in patch assays. We identified a single amino acid change at position 104 (T-A) that was responsible for the necrosis induction or loss, while a significant difference was not detected in the RSS activity of the two parental strains. We have successfully complemented the infection of the WT strain on resistant pepper cultivar with the infectious S RNA transcript of the RB strain and the WT-T104A point mutant. Our work provides direct evidence that a single amino acid change can induce an RB phenotype.

IDENTIFICATION AND MOLECULAR CHARACTERIZATION OF TOBACCO MILD GREEN MOSAIC VIRUS IN HUNGARY

Pepper (Capsicum annuum L.), an important crop in Hun- gary, is frequently infected by Cucumber mosaic virus (CMV), Tobacco mosaic virus (TMV) and, recently, also by Pepper mild mottle virus (PMMoV) and Tomato spotted wilt virus (TSWV). In the spring of 2015, mild mosaic symptoms were observed on cv. Ho F1. Symptoms on the fruits were more obvious, consisting of reduction in size, mottling and color changes, brown necrotic streaks and spots. To identify the putative pathogen(s), symptomatic fruits were collected and used for host range determination. Symptoms on mechani- cally inoculated Nicotiana tabacum cv. Samsun, N. tabacum cv. Xanthi-nc, Nicotiana benthamiana, Capsicum annuum cv. Albaregia (L+ gene), C. annuum cv. Feherozon (L1) and C. annuum cv. Brendon F1 (L3) suggested the presence of a tobamovirus belonging to pathotype 0 (Boukema, 1980). Moreover, total RNA was extracted from a symptomatic pepper fruit with the RNeasy plant mini kit (Qiagen, Ger- many) and used in RT-PCR using universal tobamovirus primers for the coat protein gene (Kalman et al., 2001). A products of the expected size (700 bp) was obtained, cloned into pGEM-T Easy Vector (Promega, USA) and sequenced (Biomi, Hungary). The sequence was deposited in GenBank under the accession No. KT374283. Blast analysis showed a 99% identity at nucleotide level with a Spanish isolate (P04/17, accession No. FN594859) of Tobacco mild green mosaic virus (TMGMV). To our knowledge, this is the first report of TMGMV on pepper in Hungary.

INVESTIGATION ON TOMATO SPOTTED WILT VIRUS INFECTING PEPPER PLANTS IN HUNGARY

In Hungary resurgence of Tomato spotted wilt virus (TSWV) frequently causes heavy crop losses in pepper production since the mid nineties. Management of TSWV control was first directed against the thrips (using different insecticides or plastic traps), and against weeds as host plants of the virus and the thrips. Later on Tsw resistance gene was introduced from Capsicum chinense PI 152225 and PI 159236 into different types of pepper. In 2010 and 2011 sporadically, but in 2012 more frequently a resistance breaking (RB) strain of TSWV on resistant pepper cultivars was observed in the Szentes region (South-East Hungary). The presence of a new resistance breaking strain was demonstrated by virological (test-plant, serological and RT-PCR) methods. Previously, the non-structural protein (NSs) encoded by small RNA (S RNA) of TSWV was verified as the avirulence factor for Tsw resistance, therefore we analyzed the S RNA of the Hungarian RB and wild type (WT) isolates and compared to previously analyzed TSWV strains with RB properties from different geographical origins. Phylogenetic analysis demonstrated that the different RB strains had the closest relationship with the local WT isolates and there was no conserved mutation present in all the NSs genes of RB isolates from different geographical origins. According to these results, it is concluded that the RB isolates evolved separately in geographic point of view and according to the RB mechanism. In order to find new genetic sources of resistance in Capsicum species 89 lines from Capsicum annuum, C. chinense, C. frutescens, C. chacoense, C. baccatum var. baccatum, C. baccatum var. pendulum and C. praetermissum were tested with the Hungarian TSWV-RB isolate.