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In nature, plants have evolved a myriad of preformed and induced defenses to protect themselves from microbes. Upon microbial infection, the recognition of the microbe-associated molecular patterns (MAMPs) by the pattern recognition receptors (PRRs) triggers the first stage of defense response (Dodds and Rathjen, Nat Rev Genet 11:539-548, 2010). However, in order to develop microbial delivery, effectors target PRRs for deregulating immune responses and facilitating host colonization (Thomma et al., Plant Cell 23:4-15, 2011). Here, we contribute a protocol for the screening system of Magnaporthe oryzae effectors and construct a fluorescent system to trace secretory proteins in the sheath infection samples. Using the tobacco rattle virus (TRV) system, the proteins including LysM, Chitin, Cutinase, and CFEM domains were selected and divided into two kinds according to the results of cell death induced or inhibited test in Nicotiana benthamiana. Then, candidate effectors can be deleted or overexpressed in M. oryzae. The barley or rice infection with M. oryzae, rice leaf sheath inoculation, and subcellular localization during infection can be performed to explore the functions of these effectors.

Virus-induced gene silencing (VIGS) uses recombinant viruses to knock down the expression of endogenous plant genes, allowing for rapid functional analysis without generating stable transgenic plants. The Tobacco rattle virus (TRV) is a popular vector for VIGS because it has a wide host range that includes Petunia × hybrida (petunia), and it induces minimal viral symptoms. Using reporter genes like chalcone synthase (CHS) in tandem with a gene of interest (GOI; pTRV2-PhCHS-GOI), it is possible to visually identify silenced flowers so that phenotyping is more accurate. Inoculation methods and environmental conditions need to be optimized for each host plant-virus interaction to maximize silencing efficiency. This chapter will provide detailed protocols for VIGS in petunia, with an emphasis on the investigation of flower phenotypes.

Research on gene functions in non-model tree species is hampered by a number of difficulties such as time-consuming genetic transformation protocols and extended period for the production of healthy transformed offspring, among others. Virus-induced gene silencing (VIGS) is an alternative approach to transiently knock out an endogenous gene of interest (GOI) by the introduction of viral sequences encompassing a fragment of the GOI and to exploit the posttranscriptional gene silencing (PTGS) mechanism of the plant, thus triggering silencing of the GOI. Here we describe the successful application of Tobacco rattle virus (TRV)-mediated VIGS through agroinoculation of olive plantlets. This methodology is expected to serve as a fast tracking and powerful tool enabling researchers from diversified fields to perform functional genomic analyses in the olive tree.

Potato is the world's fourth largest food crop and a vegetatively propagated model polyploid plant. To facilitate genomic studies in potato, here we describe detailed protocols to silence genes in both diploid potato Solanum bulbocastanum and tetraploid potato cultivars such as Maris Bard, Arran Pilot, Ancilla, and Serrana using tobacco rattle virus (TRV)- or potato virus X (PVX)-induced gene silencing (VIGS) system, respectively. The established VIGS system represents an efficient and powerful approach for functional analysis of genes involved in growth, development, metabolism, and responses to biotic and abiotic stresses in potato.

Virus-induced gene silencing (VIGS) has emerged as a fast and efficient reverse and forward genetics tool to study gene function in model plants as well as in agriculturally important plants. In addition, VIGS approach has been successfully used to provide insights into the role of several genes and regulators involved in plant secondary metabolism. Ashwagandha (Withania somnifera) is an important Indian medicinal plant that accumulates pharmacologically important triterpenoid steroidal lactones, which are collectively termed as withanolides. W. somnifera being a highly recalcitrant plant for genetic transformation, Tobacco rattle virus (TRV)-mediated VIGS was established by our group to facilitate understanding of withanolides' pathway. Here, we describe a detailed procedure to carry out VIGS for gene function studies in W. somnifera.

Virus-induced gene silencing (VIGS) is a transcript suppression technique that enables the functional characterization of genes in recalcitrant transformation plants. This technique consists in cloning a short fragment of a gene of interest into a viral vector, such as TRV (Tobacco rattle virus), and this viral construction is used to agro-infiltrate the plant. VIGS induces posttranscriptional gene silencing (PTGS) that results in the specific sequence degradation of target RNAs. Here we describe a VIGS protocol using the Gateway-based TRV vector for the study of genes in chili pepper plants.

Virus-induced gene silencing (VIGS) is a powerful epigenetic tool that allows in a relatively short period of time to down-regulate the expression of an endogenous gene in infected plants for either monitoring the resulting phenotype or enhancing/modifying a particular trait associated with the gene. Here, we describe the utilization of Tobacco rattle virus (TRV) as a vector for the VIGS technique in Arabidopsis plants. The unique ability of TRV to infect both somatic tissues and gametes allows deciphering the role of genes in these tissues simultaneously. As an example, we demonstrate the utilization of TRV to down-regulate the expression of AGO2 and NRPD1a genes in ovules of Arabidopsis plants in order to boost the stable transformation efficiency by floral dip.

Plant virus vectors have been developed as a result of 20 years of advances in molecular biology, which produced infectious plasmid-based clones of RNA and DNA plant viruses and viroids that could be routinely amplified in E. coli. This work built on a century of progress in virology, much of which was based on understanding the biology of Tobacco mosaic virus (TMV) (1). With the availability of the rice and Arabidopis draft genome sequences, expressed sequence tags (ESTs) and microarrays, crystal structures of many plant viruses, and public interest in non-transgenic strategies for crop improvement, the technology of plant virus gene vectors holds much promise for the coming decade (2, 3, 4). The intent of this review is to define virus-vector strategies, describe uses for improving crop health, and point out possible beneficial overlaps with veterinary and human medicine.

Knowledge of the molecular mechanisms underlying virus transmission is a prerequisite to the creation of new approaches to modulate vector competence and to reduce the multiplication of initial infection sites in seed and pollen. In this chapter the recent advances made in identifying the virus, vector and host determinants involved in transmission processes are described.

In common with all parasites, viruses must have a means of moving from one host to another in order to survive. For the majority of plant viruses, this phase involves the assistance, either actively or passively, of a motile vector. For most viruses, this vector is an arthropod but a small number of viruses have other vectors, prominent among which are soil-inhabiting nematodes which feed on plant roots. However, although few viruses are involved, they can nevertheless cause some economically significant diseases.

The plant diseases described in this handbook are caused by bacteria, fungi, nematodes, a few seed plants (such as dodder, mistletoe, and witchweed), viruses, physiological disturbances, and air and water pollutants. The classification of bacteria, fungi, and viruses is somewhat involved and is given here as a background for the specific descriptions in Chapter 3. There are many classifications of bacteria, fungi, and viruses, with numerous disagreements among mycologists, bacteriologists, virologists, and plant pathologists. Names and groups have been chosen that are widely accepted and most readily adapted to the alphabetical requirements of a reference that works like a dictionary.

Incidental to field observations on poplar plantations arabis mosaic virus (AMV) tomato black ring virus (TBRV) and tobacco rattle virus (TRV) were detected in roots but not leaves of Populus x euramericana cultivars. However, Nepo viruses were transmitted from leaves of other woody plants in which they were associated with symptoms of disease.

The processes involved in plant virus replication may include (1) passage of virus through the cell wall; (2) entry of virus or its nucleic acid into cells and then to replicative sites in cells; (3) removal of protein from nucleic acid, this being termed ‘uncoating’.

The discovery of plant root ectoparasitism by nematodes is as recent as 1951 when Christie and Perry (2) experimentally proved that a Trichodorus sp. (later named T. christiei) produces stubbyroot symptoms on the roots of beet and sweet corn. Ten years later, the genus Trichodorus again attracted much attention when, in the same year, three species (26) were shown to be vectors of tobacco rattle virus. Direct damage to plant roots, caused by an ectoparasite of the family Longidoridae (Longidorus, Paralongidorus and Xiphinema spp.),in this case Longidorus sp., was first recorded in 1954 (14). Three years later, Schindler (24) gave a detailed account of the pathogenicity of X. diversicaudatum. In 1958 it was first discovered that a nematode of the genus Xiphinema can transmit a virus disease in plants (12). This discovery considerably encouraged the search for nematode vectors of soil-borne plant viruses. To date several nematode species, all belonging to the dorylaimoid genera Trichodorus, Longidorus and Xiphinema, are established vectors of plant viruses.

Field trials were undertaken in two consecutive years with some systemic nematicides for the control of trichodorid nematodes and the transmission of tobacco rattle virus (TRV) to potatoes. In replicated plot experiments oxamyl (Vydate) at 5.6 or 7.8 kg a.i./ha, aldicarb (Temik) at 3.4 kg a.i./ha, phenamiphos (Nemacur) at 15.0 or 30.0 kg a.i./ha and dichloropropane-dichloropropene (D-D) at 224 kg/ha were applied to soil containing trichodorids carrying TRV. The plots were planted with a spraing-susceptible potato cultivar, Pentland Dell. Numbers of trichodorids were estimated, at about 6-weekly intervals throughout the growing season, by wet-sieving soil samples (200 g) taken from each treatment.

Members of the genus Trichodorus (21) are widespread in North America and Europe and have been recorded from all the main continental land masses of the world and from many islands, especially in the West Indies and in Australasia. Their economic significance was first demonstrated when Christie and Perry (20) showed that a species later described as T. christiei was responsible for damage to crop plants in the United States. They called it the stubby root nematode as a result of the symptoms it produced on host plants; this sobriquet was at first restricted to T. christiei but because similar symptoms are known to be caused by other species it is now customary to apply it to all members of the genus Trichodorus. Interest in the genus was further increased when it was shown that Trichodorus species could transmit tobacco rattle virus (84) and pea early browning virus (40). In 1973 Siddiqi (82) proposed the division of the genus Trichodorus sensu lato into two genera, Paratrichodorus and Trichodorus. In this paper we use the generic name Trichodorus to encompass both Paratrichodorus and Trichodorus sensu stricto as there seems at present to be little evidence of biological or behavioural differences between the two new genera; specific names, however, are given according to Siddiqi (82).

Corky ringspot (CRS) believed to be caused by tobacco rattle virus (TRV) is an important disease of potatoes in northeastern Florida, U.S.A. Although potato cultivars such as Pungo possessing resistance to CRS are available, other varietal characteristics such as susceptibility to bacterial wilt (caused by Pseudomonas solanacerarum E.F.Sm.), tendency to produce dark potato chips, and low yield potential in Florida’s sub-tropical climate, have limited their use. Since 1970 most potato fields in northeast Florida have been fumigated with chemicals such as DD for control of Belonolaimus longicaudatus Rau, Meloidogyne incognita (Kofoid & White) Chitwood, and Tylenchorhynchus claytoni Steiner, as well as nematodes in five other plant parasitic genera. There were reports from other sections of Florida that following soil fumigation, numbers of Trichodorus christiei Allen (the vector of TRV) on other crops sometimes increased to levels exceeding those in non-treated controls. Experiments were therefore initiated during 1970 to determine whether numbers of T. christiei would increase on potatoes following soil fumigation and whether incidence and severity of CRS would be affected by any observed increases; and, whether non-volatile carbamate and organophosphate nematicides would reduce incidence and severity of CRS.