Two Tobravirus expression vectors were evaluated for the use as a rapid screening method for anti-nutritional proteins against plant pathogens. Accumulation of green fluorescent protein (GFP) and snowdrop lectin gene (Galanthus nivalis agglutinin, LECGNA2, M55556) in Nicotiana benthamiana by Tobacco rattle virus expression vectors was characterized. Virally expressed proteins were detected in leaves (3-14 days post-inoculatiion) and roots (6-24 dpi) by UV (GFP), western blotting and tissue printing. 25 -50 ng of GNA was detected in root extracts. Cross protection was induced by TRV-GFP. Foreign genes inserted in place of TRV RNA2 non-structural genes (2b and 2c) were stably maintained over serial passages. But recombination at remaining 'cross-over' sites may occur. 2D iso-electricfocusing detected a 50-kDa GNA molecule in root and leaf extract. GNA did not confer resistance to root-knot nematodes, although gall by root-knot nematodes (mixed Meloidogyne spp. and M.javanica Crete line 17) were significantly reduced by 22% in roots infected by TRV-GNA (3.83 sqrt galls and 4.5 sqrt galls respectively) compared to virus-free roots treatment (4.94 sqrt galls, sed 0.398; p<.025 and 5.273 sqrt galls, sed 0.2403; <.003 respectively). Effects of GNA on Aulacorthum solani was delayed to the second nymph generation (N2). Mean N2 weights feeding on TRV-GNA (0.246 mg ±0.0159; p <05) and TRV-fsGNA (0.212 mg ±0.018; p<.001) infected plants were significantly smaller by 15.2% and 26.6% respectively, compared to virus-free treatments (0.290 mg ±0.014). Similar trends were detected for total nymph weights. Low toxicity was related to high quality phloem and ingestion of smaller volumes for normal development (i.e. concentration effect). Decrease in gall by the mixed Meloidogyne population and an unexpected toxicity to A solani indicated that truncated GNA was a protein with merolectin properties. The viability of this system as a rapid 'm planta' expression system is discussed.

Tobacco rattle virus (TRV) is a bipartite plant virus that infects potato tubers to produce the spraing or corky ring spot (CRS) disease of potato. TRV is primarily a soil-borne pathogen that is vectored by trichodorid nematodes. Spraing is characterized by the production of brown arcs and flecks in the tuber flesh or circular rings on its external surface. Spraing has been described as a hypersensitive response (HR). However, the genetic and biochemical nature of spraing had not been previously investigated experimentally. I have conducted studies to reveal the gene expression and the biochemical basis for spraing formation. Microarray analysis of RNA extracted from tuber-tissue showing spraing symptoms, revealed up-regulation of several defence related genes. Quantitative RT-PCR (qRT-PCR) of some of the differentially-expressed potato defence related genes was done for verification of the microarray data. Biochemical tests for cell death response reactions and staining for HR-related compounds or production of reactive oxygen species (ROS) also revealed the operation of HR-related processes in the spraing-affected tuber. Uneven distribution of the TRV RNA-1 in a spraing-symptomatic tuber also supports the notion that it’s a virus-induced HR-response. RNA-2 of TRV besides coding for the CP also carries the non-structural genes, 2b and 2c genes that are responsible for the nematode transmission of TRV. Fifteen different TRV recombinant isolates were prepared and the influence of the RNA-2 specific genes, encoded by a range of TRV-isolates, in causing infection among different cultivars of potato was also evaluated. Investigations were conducted to identify TRV-susceptible genotypes in which virus could move systemically and accumulate to a sufficient level to be useful for TRV-infection and VIGS-related studies for functional analysis of potato genes.

SANTRAUKA Potato mop-top virus, priklausantis Pomovirus genčiai ir Tobacco rattle virus, priklausantis Tobravirus genčiai – tai dirvožemiu plintantys virusai. Tai patogenai, kurie plačiai paplitę pasaulyje. Pastaruoju metu vis labiau susidomėta šiais virusais, mat jie sukelia daug žalos daugeliui augalų, tame tarpe ir labai svarbių kultūrinių augalų. Su šiais patogenais kovoti yra labai sudėtinga, taigi iškyla labai rimta problema, kurią bandoma išspręsti. Todėl šiame darbe ir buvo siekiama susipažinti su šiais virusais, jų pernešėjais bei padaryti pirmuosius žingsnius, ieškant būdus kovai su šais patogenais. PMTV – tai patogenas, kuriam nebūdingas gausus šeimininkų ratas. Jis infekuoja tik nedaugelį augalų rūšių. Tai viena iš savybių, kuria skiriasi nuo TRV. Pastarasis priešingai, pasižymi didele augalų-šeimininkų gausa. Šie virusai labai panašūs tuom, kad daugelyje augalų jie sukelia panašius simptomus bei pažeidimus. Net indikatoriniuose augaluose jų sukeltos žaizdos yra labai panašios. Taigi, susiduriama su problema, mat identifikuoti juos vizualiai yra labai sudėtinga. Tik N. benthamiana indikatorinis augalas gali padėti atskirti šiuos virusus: PMTV jame sukelia sisteminę reakciją, sisteminė mozaika išplinta po visą augalą, o TRV sukelia tik vietines žaizdas. Šiame baigiamajame darbe buvo siekiama identifikuoti PMTV bei TRV molekuliniais metodais. Identifikacijoje buvo pritaikytas imunofermentinis metodas – ELISA. Buvo nustatyta, kad šis metodas labiau tinka... [toliau žr. visą tekstą] / SUMMARY Potato mop-top virus (genus Pomovirus) and Tobacco rattle virus (genus Tobravirus) – soil born viruses. These pathogenes are distributed worldwide and can significantly reduce the quality and yield of potato and other horticultural plants. So in recent years more and more people are interesting in these viruses. These viruses are responsible for economic losses in potato crops. To control the spread of viruses is vary difficult, so nowadays it is a serious problem, which everybody wants to solve it. Virus control depends on vector management, so the main purpose of this article was to know more about these viruses and thier vectors and to do first steps to finding ways of viruses’ suitable control measures. PMTV has narrow range of hosts. On the contrary TRV has a very wide natural host range. So it is one of the differences from these soil borne viruses. However diagnosis is further complicated by the fact that TRV and PMTV are very similar viruses, even they can cause similar symptoms to indicator plants. So to diagnose reliable these viruses by visual symptoms are very difficult. Just in indicator plant N. benthamiana these soil borne viruses cause different symptoms: PMTV cause sistemical reaction, sistemical mosaic spreads in whole plant and TRV cause local lessions. PMTV and TRV were identificated with different molecular methods. These viruses were detected by enzyme-linked immunosorbent assay. DAS-ELISA was shown to be more sensetive and reliable method for... [to full text]

Résumé : Pour se propager dans les cellules de son hôte et évader les réponses immunitaires, les virus végétaux ont développé plusieurs stratégies de défense. Ici, nous avons investigué les structures génétiques du Apple stem pitting virus (ASPV). Nous avons aussi étudié la diversité moléculaire des isolats d’ASPV provenant des poires en regardant les séquences des gènes CP et TGB afin de mieux comprendre les mécanismes évolutionnaires utilisés par ASPV. Nos études ont démontré que les mutations, incluant les insertions et les délétions, la sélection purificatrice et la recombinaison furent des facteurs importants dans l’évolution du l’ASPV en Chine et possiblement mondialement. Comme tous les virus végétaux, l’ASPV se défend contre le RNA silencing de l’hôte grâce à un suppresseur de RNA silencing (VSR) et nous avons montré que le VSR de l’ASPV est la protéine de capside (CP) du virus. Nous avons aussi établi que la diversité moléculaire cause non seulement une variété de symptômes chez son hôte, Nicotiana occidentalis. Cependant elle cause aussi de la variabilité antigénique chez différents isolats, ce qui mène à des écarts de réactivité sérologique entre isolats. Les plantes ont développé plusieurs stratégies pour se défendre contre les virus. Ici, nous avons étudié comment la plante Arabidopsis se défend contre le Tobacco rattle virus (TRV) via le RNA silencing. Nous avons constaté que les phénomènes de susceptibilité, récupération et virus induced gene silencing (VIGS) sont des mécanismes séparables. Nous avons démontré que les protéines AGO2 et AGO4 sont nécessaires à la susceptibilité initiale au TRV, tandis qu’AGO1 est importante pour les VIGS, tandis que la récupération est médiée par d’autres acteurs qui n’ont pas encore été identifiés. Nos résultats suggèrent l’existence de complexes distincts ciblant différentes populations d’ARN viral et cellulaire. De plus, nous avons montré que la répression de la traduction est un mécanisme important durant la récupération de la plante suite à une infection virale, et que les complexes de décoiffage et de RNA processing jouent des rôles importants dans la dégradation des ARNs viraux. Finalement, nous avons montré que les plantes ayant une mutation dans le gène DCP2 présentent un niveaux de VIGS accrue, ainsi qu’une augmentation des niveaux d’ARN viral. Puisque DCP2 fait partie des complexes de décoiffage qui se trouvent dans des granules spécialisés nommés processing bodies (PBs), cela suggère que les PBs jouent un rôle important dans l’élimination les virus. / Abstract : To live in host cells or to escape from host immunity, plant viruses involved a series of defense strategies. Here we investigated Apple stem pitting virus (ASPV) population structures and molecular diversity of ASPV pear isolates based on its function important gene CP and TGB in China, so as to infer the evolution mechanisms of ASPV. Our study showed that mutations (including insertions or deletions), purifying selection, and recombination were important factors driving ASPV evolutions in China or maybe even in the world. And also ASPV defends against it hosts by encoding a VSR. We also showed that ASPV molecular diversity not only induced different biological properties on its herbaceous host N. occidentails but also resulted in antigenic variation of different ASPV CP isolates, which leaded to differences in serological reactivity among rCPs of different ASPV isolates. Plants have developed a series of mechanisms to defend themselves against viruses. Here we how Arabidopsis defend against. We show that virus susceptibility, recovery, and virus induced gene silencing (VIGS) appear to be separable phenomena, with AGO2 and AGO4 playing important roles in the initial susceptibility to TRV, AGO1 playing an important role in VIGS, and as yet unidentifid players mediating recovery. These results suggest the existence of distinct RNA-induced silencing complexes that target different RNA populations within the cell and over time. Furthermore, we showed that translational repression of viral RNA is likely to play an important role in virus recovery and that decapping function plays an important role in clearing viral RNA from the cell. We also showed that a decapping mutant (DCP2) displayed an increased VIGS and virus RNA accumulation, an important role for PBs in eliminating viral RNA.

The current study focuses on the vascular cambium and the reiterative formation of phloem fiber bundles in cotton stems. The role of the TDIF-PXY-WOX pathway was examined in regulating cambial activity and the differentiation of phloem fibers. A study was conducted to identify and characterize the cotton WOX family genes, focusing on WOX4 and WOX14, aiming to identify and analyze their phylogenetic relationships, tissue-specific expression profiles, functional roles, and metabolic consequences. Through a sequence analysis of the Gossypium hirsutum genome, 42 cotton loci were identified as WOX family members. GhWOX4 exhibited a close homology to 7 loci, while GhWOX14 displayed homology with 8 loci. Tissue-specific expression analysis revealed prominent expression patterns of GhWOX4 and GhWOX14 in cotton internodes and roots, suggesting their involvement in vascular tissue development. Functional studies utilizing VIGS (virus-induced gene silencing) demonstrated that the knockdown of GhWOX4 and GhWOX14 resulted in a significant reduction in stem diameter and bast fiber production. This result suggests that secondary phloem fiber development is regulated by GhWOX4 and GhWOX14 genes in cotton. Additionally, the metabolic profiling of VIGS plants revealed significant alterations in amino acids, organic acids, and sugars, with implications for primary metabolic pathways. These findings suggest that GhWOX4 and GhWOX14 play pivotal roles in cotton plant development, including vascular tissue growth and phloem fiber production, and metabolic regulation.

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