Development and Application of Attenuated Plant Viruses as Biological Control Agents in Japan.

In 1929, it was reported that yellowing symptoms caused by a tobacco mosaic virus (TMV) yellow mosaic isolate were suppressed in tobacco plants that were systemically infected with a TMV light green isolate. Similar to vaccination, the phenomenon of cross-protection involves a whole plant being infected with an attenuated virus and involves the same or a closely related virus species. Therefore, attenuated viruses function as biological control agents. In Japan, many studies have been performed on cross-protection. For example, the tomato mosaic virus (ToMV)-L11A strain is an attenuated isolate developed by researchers and shows high control efficiency against wild-type ToMV in commercial tomato crops. Recently, an attenuated isolate of zucchini yellow mosaic virus (ZYMV)-2002 was developed and registered as a biological pesticide to control cucumber mosaic disease. In addition, attenuated isolates of pepper mild mottle virus (PMMoV), cucumber mosaic virus (CMV), tobacco mild green mosaic virus (TMGMV), melon yellow spot virus (MYSV), and watermelon mosaic virus (WMV) have been developed in Japan. These attenuated viruses, sometimes called plant vaccines, can be used not only as single vaccines but also as multiple vaccines. In this review, we provide an overview of studies on attenuated plant viruses developed in Japan. We also discuss the application of the attenuated strains, including the production of vaccinated seedlings.

Identification of a novel viral factor inducing tumorous symptoms by disturbing vascular development in planta.

Plant viruses induce various disease symptoms that substantially impact agriculture, but the underlying mechanisms of viral disease in plants are poorly understood. Kobu-sho is a disease in gentian that shows gall formation with ectopic development of lignified cells and vascular tissues such as xylem. Here, we show that a gene fragment of gentian Kobu-sho-associated virus, which is designated as Kobu-sho-inducing factor (KOBU), induces gall formation accompanied by ectopic development of lignified cells and xylem-like tissue in Nicotiana benthamiana. Transgenic gentian expressing KOBU exhibited tumorous symptoms, confirming the gall-forming activity of KOBU. Surprisingly, KOBU expression can also induce differentiation of an additional leaf-like tissue on the abaxial side of veins in normal N. benthamiana and gentian leaves. Transcriptome analysis with Arabidopsis thaliana expressing KOBU revealed that KOBU activates signaling pathways that regulate xylem development. KOBU protein forms granules and plate-like structures and co-localizes with mRNA splicing factors within the nucleus. Our findings suggest that KOBU is a novel pleiotropic virulence factor that stimulates vascular and leaf development. IMPORTANCE While various mechanisms determine disease symptoms in plants depending on virus-host combinations, the details of how plant viruses induce symptoms remain largely unknown in most plant species. Kobu-sho is a disease in gentian that shows gall formation with ectopic development of lignified cells and vascular tissues such as xylem. Our findings demonstrate that a gene fragment of gentian Kobu-sho-associated virus (GKaV), which is designated as Kobu-sho-inducing factor, induces the gall formation accompanied by the ectopic development of lignified cells and xylem-like tissue in Nicotiana benthamiana. The molecular mechanism by which gentian Kobu-sho-associated virus induces the Kobu-sho symptoms will provide new insight into not only plant-virus interactions but also the regulatory mechanisms underlying vascular and leaf development.

How do emerging long-read sequencing technologies function in transforming the plant pathology research landscape?

KEY MESSAGE: Long-read sequencing technologies are revolutionizing the sequencing and analysis of plant and pathogen genomes and transcriptomes, as well as contributing to emerging areas of interest in plant-pathogen interactions, disease management techniques, and the introduction of new plant varieties or cultivars. Long-read sequencing (LRS) technologies are progressively being implemented to study plants and pathogens of agricultural importance, which have substantial economic effects. The variability and complexity of the genome and transcriptome affect plant growth, development and pathogen responses. Overcoming the limitations of second-generation sequencing, LRS technology has significantly increased the length of a single contiguous read from a few hundred to millions of base pairs. Because of the longer read lengths, new analysis methods and tools have been developed for plant and pathogen genomics and transcriptomics. LRS technologies enable faster, more efficient, and high-throughput ultralong reads, allowing direct sequencing of genomes that would be impossible or difficult to investigate using short-read sequencing approaches. These benefits include genome assembly in repetitive areas, creating more comprehensive and exact genome determinations, assembling full-length transcripts, and detecting DNA and RNA alterations. Furthermore, these technologies allow for the identification of transcriptome diversity, significant structural variation analysis, and direct epigenetic mark detection in plant and pathogen genomic regions. LRS in plant pathology is found efficient for identifying and characterization of effectors in plants as well as known and unknown plant pathogens. In this review, we investigate how these technologies are transforming the landscape of determination and characterization of plant and pathogen genomes and transcriptomes efficiently and accurately. Moreover, we highlight potential areas of interest offered by LRS technologies for future study into plant-pathogen interactions, disease control strategies, and the development of new plant varieties or cultivars.

A novel FLOURY ENDOSPERM2 (FLO2)-interacting protein, is involved in maintaining fertility and seed quality in rice.

Crop plants accumulate a large amount of storage starch and storage proteins in the endosperm. Genes involved in the biosynthesis of these substances work in concert during development of the rice endosperm. The rice flo2 mutant produces aberrant seeds with reduced grain quality. FLOURRY ENDOSPERM 2 (FLO2), the causative gene of the flo2 mutant, is considered to be a regulatory protein that controls the biosynthesis of seed storage substances. FLO2 contains tetratricopeptide repeat (TPR) motifs that may mediate protein-protein interactions. In this study, we identified the protein that interacts with the TPR motif of FLO2. We generated a transformant that produced the FLAG-tagged fusion FLO2 protein in the flo2 mutant and used this in the shotgun proteomic analysis. A protein, which we named FLOC1, interacted with FLO2. In vitro pull-down assays indicated that the TPR motif was involved in this interaction. A knock-down transformant of FLOC1 showed significantly reducted fertility and generation of seeds with abnormal features. These findings suggest that FLOC1 is involved not only in seed fertility but also in seed quality. These phenotypes were also observed on the RNAi transformants of the flo2 mutant although the effect of the flo2 mutation remained. these findings imply that there is a difference in the functions of FLO2 and FLOC1 although both of appear to be involved in the control of seed quality during seed formation.

Identification and Functional Analysis of NB-LRR-Type Virus Resistance Genes: Overview and Functional Analysis of Candidate Genes.

Coexpression of a plant NB-LRR-type resistance (R) gene and corresponding viral avirulent (Avr) gene introduced in Nicotiana benthamiana using Agrobacterium tumefaciens confers hypersensitive response (HR). Such Agrobacterium-mediated transient gene expression methods have contributed to the identification of new plant R genes and facilitated the analysis of their functions. Here we describe a model method, by which several tobamovirus R genes from Solanaceous plants have been successfully identified and characterized molecularly.

Insertion of Epitope Tag into NB-LRR Class Plant Virus Resistance Protein.

It is prerequisite to detect plant disease resistance proteins for studying the function of the proteins. Numerous studies have used epitope tags fused to either N- or C-terminus for the detection of resistance proteins. However, some resistance proteins do not tolerate the terminal fusions of epitope tags. In this chapter, we provide a protocol for searching the protein regions in which the inserted epitope tag does not affect the protein function. In the protocol, we first perform an in silico search to select the insertion site candidates and then insert there a short sequence containing restriction sites to find out the sites, in which the insertion does not affect the protein function. Epitope tags are inserted into the experimentally selected sites to produce a functional protein with an epitope tag.

Random Mutagenesis of Virus Gene for the Experimental Evaluation of the Durability of NB-LRR Class Plant Virus Resistance Gene.

NB-LRR class plant virus resistance gene is a one of the key players that shape the plant-virus interaction. Evolutionary arms race between plants and viruses often results in the breakdown of virus resistance in plants, which leads to a disastrous outcome in agricultural production. Although studies have analyzed the nature of plant virus resistance breakdown, it is still difficult to foresee the breakdown of a given virus resistance gene. In this chapter, we provide a protocol for evaluating the durability of plant virus resistance gene, which comprises the random mutagenesis of a virus gene, the introduction of the mutagenized gene into a virus context with highly efficient inoculation system, and the efficient screening of virus mutants that can overcome or escape a virus resistance.

The Host Stomatal Density Determines Resistance to Septoria gentianae in Japanese Gentian.

Plant stomata represent the main battlefield for host plants and the pathogens that enter plant tissues via stomata. Septoria spp., a group of ascomycete fungi, use host plant stomata for invasion and cause serious damage to agricultural plants. There is no evidence, however, showing the involvement of stomata in defense systems against Septoria infection. In this study, we isolated Septoria gentianae 20-35 (Sg20-35) from Gentiana triflora showing gentian leaf blight disease symptoms in the field. Establishment of an infection system using gentian plants cultured in vitro enabled us to observe the Sg20-35 infection process and estimate its virulence in several gentian cultivars or lines. Sg20-35 also entered gentian tissues via stomata and showed increased virulence in G. triflora compared with G. scabra and their interspecific hybrid. Notably, the susceptibility of gentian cultivars to Sg20-35 was associated with their stomatal density on the adaxial but not abaxial leaf surface. Treatment of EPIDERMAL PATTERNING FACTOR-LIKE 9 (EPFL9/STOMAGEN) peptides, a small secreted peptide controlling stomatal density in Arabidopsis thaliana, increased stomatal density on the adaxial side of gentian leaves as well. Consequently, treated plants showed enhanced susceptibility to Sg20-35. These results indicate that stomatal density on the adaxial leaf surface is one of the major factors determining the susceptibility of gentian cultivars to S. gentianae and suggest that stomatal density control may represent an effective strategy to confer Septoria resistance.

Efficacy of a Simple Formulation Composed of Nematode-Trapping Fungi and Bidens pilosa var. radiata Scherff Aqueous Extracts (BPE) for Controlling the Southern Root-Knot Nematode.

We tested a formulation composed of a mixture of Bidens pilosa var. radiata extract (BPE) and nematode-trapping fungi for its effects on Meloidogyne incognita. In earlier evaluations of the effects of plant extracts on the hyphal growth of 5 species of nematode-trapping fungi with different capture organs (traps), the growth of all species was slightly inhibited. However, an investigation on the number of capture organs and nematode-trapping rates revealed that Arthrobotrys dactyloides formed significantly more rings and nematode traps than those of the control. An evaluation of simple mixed formulations prepared using sodium alginate showed that nematodes were captured with all formulations tested. The simple mixed formulation showed a particularly high capture rate. Furthermore, in a pot test, although the effects of a single formulation made from the fungus or plant extract were acceptable, the efficacy of the simple mixed formulation against M. incognita root-knot formation was particularly high.

Inducible expression of magnesium protoporphyrin chelatase subunit I (CHLI)-amiRNA provides insights into cucumber mosaic virus Y satellite RNA-induced chlorosis symptoms.

Recent studies with Y satellite RNA (Y-sat) of cucumber mosaic virus have demonstrated that Y-sat modifies the disease symptoms in specific host plants through the silencing of the magnesium protoporphyrin chelatase I subunit (CHLI), which is directed by the Y-sat derived siRNA. Along with the development of peculiar yellow phenotypes, a drastic decrease in CHLI-transcripts and a higher accumulation of Y-sat derived siRNA were observed. To investigate the molecular mechanisms underlying the Y-sat-induced chlorosis, especially whether or not the reduced expression of CHLI causes the chlorosis simply through the reduced production of chlorophyll or it triggers some other mechanisms leading to the chlorosis, we have established a new experimental system with an inducible silencing mechanism. This system involves the expression of artificial microRNAs targeting of Nicotiana tabacum CHLI gene under the control of chemically inducible promoter. The CHLI mRNA levels and total chlorophyll content decreased significantly in 2 days, enabling us to analyze early events in induced chlorosis and temporary changes therein. This study revealed that the silencing of CHLI did not only result in the decreased chlorophyll content but also lead to the downregulation of chloroplast and photosynthesis-related genes expression and the upregulation of defense-related genes. Based on these results, we propose that the reduced expression of CHLI could activate unidentified signaling pathways that lead plants to chlorosis.

Inducible transgenic tobacco system to study the mechanisms underlying chlorosis mediated by the silencing of chloroplast heat shock protein 90.

Chlorosis is one of the most common symptoms of plant diseases, including those caused by viruses and viroids. Recently, a study has shown that Peach latent mosaic viroid (PLMVd) exploits host RNA silencing machinery to modulate the virus disease symptoms through the silencing of chloroplast-targeted heat shock protein 90 (Hsp90C). To understand the molecular mechanisms of chlorosis in this viroid disease, we established an experimental system suitable for studying the mechanism underlying the chlorosis induced by the RNA silencing of Hsp90C in transgenic tobacco. Hairpin RNA of the Hsp90C-specific region was expressed under the control of a dexamethasone-inducible promoter, resulted in the silencing of Hsp90C gene in 2 days and the chlorosis along with growth suppression phenotypes. Time course study suggests that a sign of chlorosis can be monitored as early as 2 days, suggesting that this experimental model is suitable for studying the molecular events taken place before and after the onset of chlorosis. During the early phase of chlorosis development, the chloroplast- and photosynthesis-related genes were downregulated. It should be noted that some pathogenesis related genes were upregulated during the early phase of chlorosis in spite of the absence of any pathogen-derived molecules in this system.

Horizontal pollen transmission of Gentian ovary ring-spot virus is initiated during penetration of the stigma and style by infected pollen tubes.

Gentian ovary ring-spot virus (GORV) infected gentian plants by pollination with GORV-infected gentian pollen grains, but the virus was not horizontally transmitted to gentian plants by transfer of pollen from GORV-infected Nicotiana benthamiana plants. However, N. benthamiana plants were infected with the virus by pollination with infected gentian pollen as well as by pollination with infected N. benthamiana pollen. When infected gentian pollen grains were placed on N. benthamiana stigmas, germinating pollen tubes penetrated into the stigmas and the styles (stigma-style). Virus infection occurred during penetration of the stigma-style, and the virus subsequently spread systemically to the mother plant. On the other hand, most infected N. benthamiana pollen grains failed to germinate on gentian stigmas, and virus infections were not detected in the stigma-style.

Review of Beet pseudoyellows virus genome structure built the consensus genome organization of cucumber strains and highlighted the unique feature of strawberry strain.

The complete nucleotide sequences of Beet pseudoyellows virus (BPYV)-MI (cucumber isolate; Matsuyama, Idai) genomic RNAs 1 and 2 were determined and compared with the previously sequenced Japanese cucumber strain (BPYV-JC) and a strawberry strain (BPYV-S). The RNA 2 of BPYV-MI showed 99 % nucleotide sequence identity with both BPYV-JC and -S having highly conserved eight ORFs. In contrast, the RNA1 of BPYV-MI showed sequence identities of 98 and 86 % with BPYV-JC and -S, respectively. Phylogenetic analysis of RNA-dependent RNA polymerase (RdRp) coding sequences from three fully sequenced BPYV strains and five partially sequenced cucurbit-infecting BPYV strains from Japan and South Africa has shown that cucurbit-infecting strains are closer to each other than to BPYV-S. In addition, the strawberry strain BPYV-S has an ORF2 in the downstream of RdRp gene in RNA1, but all the cucumber strains, BPYV-JC, -MI, and those from South Africa, lacked the ORF2 of RNA1, highlighting the difference between common BPYV cucumber strains and a unique strawberry strain.

Combined DECS Analysis and Next-Generation Sequencing Enable Efficient Detection of Novel Plant RNA Viruses.

The presence of high molecular weight double-stranded RNA (dsRNA) within plant cells is an indicator of infection with RNA viruses as these possess genomic or replicative dsRNA. DECS (dsRNA isolation, exhaustive amplification, cloning, and sequencing) analysis has been shown to be capable of detecting unknown viruses. We postulated that a combination of DECS analysis and next-generation sequencing (NGS) would improve detection efficiency and usability of the technique. Here, we describe a model case in which we efficiently detected the presumed genome sequence of Blueberry shoestring virus (BSSV), a member of the genus Sobemovirus, which has not so far been reported. dsRNAs were isolated from BSSV-infected blueberry plants using the dsRNA-binding protein, reverse-transcribed, amplified, and sequenced using NGS. A contig of 4,020 nucleotides (nt) that shared similarities with sequences from other Sobemovirus species was obtained as a candidate of the BSSV genomic sequence. Reverse transcription (RT)-PCR primer sets based on sequences from this contig enabled the detection of BSSV in all BSSV-infected plants tested but not in healthy controls. A recombinant protein encoded by the putative coat protein gene was bound by the BSSV-antibody, indicating that the candidate sequence was that of BSSV itself. Our results suggest that a combination of DECS analysis and NGS, designated here as "DECS-C," is a powerful method for detecting novel plant viruses.

The Chloroplastic Protein THF1 Interacts with the Coiled-Coil Domain of the Disease Resistance Protein N' and Regulates Light-Dependent Cell Death.

One branch of plant immunity is mediated through nucleotide-binding/Leu-rich repeat (NB-LRR) family proteins that recognize specific effectors encoded by pathogens. Members of the I2-like family constitute a well-conserved subgroup of NB-LRRs from Solanaceae possessing a coiled-coil (CC) domain at their N termini. We show here that the CC domains of several I2-like proteins are able to induce a hypersensitive response (HR), a form of programmed cell death associated with disease resistance. Using yeast two-hybrid screens, we identified the chloroplastic protein Thylakoid Formation1 (THF1) as an interacting partner for several I2-like CC domains. Co-immunoprecipitations and bimolecular fluorescence complementation assays confirmed that THF1 and I2-like CC domains interact in planta and that these interactions take place in the cytosol. Several HR-inducing I2-like CC domains have a negative effect on the accumulation of THF1, suggesting that the latter is destabilized by active CC domains. To confirm this model, we investigated N', which recognizes the coat protein of most Tobamoviruses, as a prototypical member of the I2-like family. Transient expression and gene silencing data indicated that THF1 functions as a negative regulator of cell death and that activation of full-length N' results in the destabilization of THF1. Consistent with the known function of THF1 in maintaining chloroplast homeostasis, we show that the HR induced by N' is light-dependent. Together, our results define, to our knowledge, novel molecular mechanisms linking light and chloroplasts to the induction of cell death by a subgroup of NB-LRR proteins.

Production of recombinant thanatin in watery rice seeds that lack an accumulation of storage starch and proteins.

Molecular farming is a promising method for producing materials of commercial interest. Plants can be expected to be appropriate hosts for recombinant protein production. However, production in genetically modified plants has two major challenges that must be resolved before its practical use: insufficient accumulation of products and difficulty in establishing methods for their purification. We propose a simple procedure for the production of a desired protein using watery rice seeds lacking an accumulation of storage starch and proteins, a phenotype induced by the introduction of an antisense SPK. We produced a transgenic rice plant containing a gene for an antimicrobial peptide, thanatin, together with antisense SPK. Bioassay and proteome analysis indicated that recombinant thanatin accumulated in an active form in these watery rice seeds. These results suggest that our system worked effectively for the production of thanatin. This procedure enabled easy removal of impurities and simplified the purification process compared with production in leaves. Our system may therefore be a useful technique for the production of desired materials, including proteins.

A new method to isolate total dsRNA.

When a diseased plant is suspected to be infected with unknown viruses, the approach of isolating double-stranded RNA (dsRNA) from diseased tissues and analyzing the sequence has been useful for detecting the viruses. This procedure owes its success to the majority of plant pathogenic viruses being RNA viruses, which accumulate dsRNAs as copies of their genome or as a replicative intermediate in infected cells. Conventional dsRNA isolation methods (e.g., chromatography using CF-11 cellulose) require a significant amount of plant material and are laborious and time consuming. Therefore, it has been impractical to isolate dsRNA from many samples at the same time. To overcome these problems, we developed a novel dsRNA isolation method involving a recombinant dsRNA-binding protein. Using this method, we can readily isolate viral dsRNA from a small amount of plant material, and can process numerous samples simultaneously. Purified dsRNA can be used as a template for cDNA synthesis and sequencing, enabling detection of both known and unknown viruses.

A novel virus transmitted through pollination causes ring-spot disease on gentian (Gentiana triflora) ovaries.

In this study, we identified a novel virus from gentian (Gentiana triflora) that causes ring-spots on ovaries. Furthermore, the virus causes unusual symptoms, ring-spots that appear specifically on the outer surface of the ovarian wall after pollination. Pollen grains carrying the virus were used to infect host plants by hand-pollination. RNA extracted from purified virions indicated that the virus had two segments, RNA1 and RNA2. The full-length cDNA sequence indicated that RNA1 had two ORFs: ORF1 had methyltransferase and helicase motifs, and ORF2 had an RNA-dependent RNA polymerase motif. RNA2 had five ORFs encoding a coat protein, triple gene block proteins 1-3 and a cysteine-rich protein. The length of RNA1 was 5519 bases and that of RNA2 was 3810 bases not including a polyU/polyA region between the first and second ORFs. Viral RNA does not have a polyA tail at the 3' end. Sequence similarity and phylogenetic analysis suggested that the virus is closely related to members of the genera Pecluvirus and Hordeivirus but distinct from them. These combined results suggest that the causal agent inducing ring-spot symptoms on gentian ovaries is a new virus belonging to the family Virgaviridae but not to any presently known genus. We tentatively name the virus gentian ovary ring-spot virus.

Prevalence and genetic diversity of an unusual virus associated with Kobu-sho disease of gentian in Japan.

Gentian Kobu-sho-associated virus (GKaV) is a recently discovered novel virus from Kobu-sho (a hyperplastic or tumorous disorder)-affected Japanese gentians. To obtain insight into GKaV transmission and pathogenesis, the genetic diversity of the virus in the putative helicase and RNA-dependent RNA polymerase coding regions was studied. The extent of GKaV sequence diversity within single host plants differed within samples and between viral genomic regions. Phylogenetic analysis of 30 Kobu-sho-affected samples from different production areas and host cultivars revealed that GKaV populations have diverged as they became prevalent in different geographical regions. The diversification of GKaV was shown to be driven by geographical isolation rather than host adaptation; however, no geographical patterns were found. Therefore, it was not feasible to trace the pathway of GKaV spread.

Establishment of an agroinoculation system for broad bean wilt virus 2.

We determined the complete nucleotide sequence of a broad bean wilt virus 2 (BBWV-2) isolate from gentian in Japan. The full-length RNA1 and RNA2 sequences, excluding poly(A) tails, were 5955 and 3600 nucleotides long, respectively. Analysis indicated that, in contrast to other BBWV-2 isolates, the 5' end of both RNA1 and RNA2 starts with a GUU sequence. We successfully inoculated Nicotiana benthamiana with BBWV-2 by infiltrating a mixed suspension of two Agrobacterium tumefaciens clones carrying binary vectors with the full-length RNA1 and RNA2 sequences. This is the first report on the efficient, easy and high-throughput use of agroinoculation for generating BBWV-2 infections.