Cassava mosaic disease and its management in Southeast Asia.

KEY MESSAGE: Status of the current outbreak of cassava mosaic disease (CMD) in Southeast Asia was reviewed. Healthy cassava seed production and dissemination systems have been established in Vietnam and Cambodia, along with integrated disease and pest management systems, to combat the outbreak. Cassava (Manihot esculenta Crantz) is one of the most important edible crops in tropical and subtropical regions. Recently, invasive insect pests and diseases have resulted in serious losses to cassava in Southeast Asia. In this review we discuss the current outbreak of cassava mosaic disease (CMD) caused by the Sri Lankan cassava mosaic virus (SLCMV) in Southeast Asia, and summarize similarities between SLCMV and other cassava mosaic begomoviruses. A SATREPS (Science and Technology Research Partnership for Sustainable Development) project "Development and dissemination of sustainable production systems based on invasive pest management of cassava in Vietnam, Cambodia and Thailand", was launched in 2016, which has been funded by The Japan International Cooperation Agency (JICA) and The Japan Science and Technology Agency (JST), Japan. The objectives of SATREPS were to establish healthy seed production and dissemination systems for cassava in south Vietnam and Cambodia, and to develop management systems for plant diseases and insect pests of cassava. To achieve these goals, model systems of healthy seed production in Vietnam and Cambodia have been developed incorporating CMD-resistant planting materials through international networks with The International Center for Tropical Agriculture (CIAT) and The International Institute of Tropical Agriculture (IITA).

Detection of Sri Lankan cassava mosaic virus by loop-mediated isothermal amplification using dried reagents.

Recently, the widespread occurrence of Sri Lankan cassava mosaic virus (SLCMV), genus Begomovirus, family Geminiviridae, which causes a mosaic disease in cassava (Manihot esculenta Crantz) in South-East Asia have, become a serious economic issue. Since cassava is propagated through vegetative cuttings, a rapid virus diagnostic method is crucial for generating virus-free planting materials. In this study, a loop-mediated isothermal amplification (LAMP) assay using six primers was developed and validated for the rapid detection of SLCMV in cassava leaves. This SLCMV assay had a detection sensitivity that was up to 10,000 times higher than that of the conventional polymerase chain reaction assay and can detect the virus from symptomless stem cutting, which is a potential long-distance spreader of the virus. Furthermore, a practical LAMP protocol using stable dried reagents from a commercial kit was established so that the assay could be performed in the field by incubating the reactions in water at 60-65 °C instead of using a thermal cycler. The primer sequences and the LAMP protocol described here should be useful for the rapid and sensitive on-site detection of SLCMV.

Mastrevirus Rep and RepA Proteins Suppress de novo Transcriptional Gene Silencing.

Transcriptional gene silencing (TGS) in plants is a defense mechanism against DNA virus infection. The genomes of viruses in the Geminiviridae family encode several TGS suppressors. In this study, we induced de novo TGS against the transgenic GFP gene encoding green fluorescent protein by expressing a hairpin-shaped self-complementary RNA corresponding to the enhancer region of the 35S promoter (hpE35S). In addition, we examined the TGS suppression activity of proteins encoded in the genome of Tobacco yellow dwarf virus (TYDV, genus Mastrevirus). The results show that the replication-associated protein (Rep) and RepA encoded by TYDV have TGS suppressor activity and lead to decreased accumulation of 24-nt siRNAs. These results suggest that Rep and RepA can block the steps before the loading of siRNAs into Argonaute (AGO) proteins. This is the first report of TGS suppressors in the genus Mastrevirus.

The genome of the Cauliflower mosaic virus, a plant pararetrovirus, is highly methylated in the nucleus.

Cytosine methylation is an important defense against invasive DNAs. Here, cytosine methylation profiles of a plant pararetrovirus, Cauliflower mosaic virus (CaMV), were investigated. Nuclear CaMV DNA is highly methylated throughout the genome including at transcription regulatory regions, but the virion DNA is unmethylated. In vitro CG methylation of the viral 35S promoter reduces transcription from the downstream gene. Although nuclear CaMV DNA is highly methylated, its transcripts are accumulated in the nucleus. The data suggest that a small population of unmethylated viral genomes produced through reverse transcription are constantly delivered back to the nucleus. Small RNA profiles suggest that methylation of the CaMV DNA may be due to de novo methylation through 21-, 22-, and 24-nt small RNAs with adenines at their 5' terminus.

Complete Genome Sequences of Seven Peanut Stunt Virus Strains from Japan.

We present the complete genomic RNA sequences of seven isolates of Peanut stunt virus discovered in diseased legume plants in various regions in Japan. These sequences and the published viral sequences were compared with respect to nucleotide percentages. Their phylogenetic analysis showed that all the isolates belong to subgroup IA.

Complete Genome Sequences of Three Tomato Aspermy Virus Isolates in Japan.

We report here the first complete nucleotide sequences of genomic RNAs of three Tomato aspermy virus (TAV) isolates in Japan. Analysis of these sequences showed that they have unique characteristics in RNAs 2 and 3. The Japanese isolates are similar to each other compared to other TAVs.

DDM1 (decrease in DNA methylation) genes in rice (Oryza sativa).

Regulation of cytosine methylation in the plant genome is of pivotal in determining the epigenetic states of chromosome regions. Relative tolerance of plant to deficiency in cytosine methylation provides unparalleled opportunities to study the mechanism for regulation of cytosine methylation. The Decrease in DNA Methylation 1 (DDM1) of Arabidopsis thaliana is one of the best characterized plant epigenetic regulators that are necessary for maintenance of cytosine methylation in genomic DNA. Although cytosine methylation could affect various aspects of plant growth and development including those related to agricultural importance, orthologs of DDM1 in plants other than Arabidopsis has not been studied in detail. In this study, we identified two rice genes with similarity to Arabidopsis DDM1 and designated them OsDDM1a and OsDDM1b. Both of the rice DDM1 homologs are transcribed during development and their amino acid sequences are 93 % identical to each other. Transgenic rice lines expressing the OsDDM1a cDNA in the antisense orientation exhibited genomic DNA hypomethylation. In those lines, repeated sequences were more severely affected than a single copy sequence as is the case in Arabidopsis ddm1 mutants. Transcripts derived from endogenous transposon-related loci were up-regulated in the antisense OsDDM1 lines, opening a possibility to identify and utilize potentially active transposons for rice functional genomics.

Pseudo-polyprotein translated from the full-length ORF1 of capillovirus is important for pathogenicity, but a truncated ORF1 protein without variable and CP regions is sufficient for replication.

The first open-reading frame (ORF) of the genus Capillovirus encodes an apparently chimeric polyprotein containing conserved regions for replicase (Rep) and coat protein (CP), while other viruses in the family Flexiviridae have separate ORFs encoding these proteins. To investigate the role of the full-length ORF1 polyprotein of capillovirus, we generated truncation mutants of ORF1 of apple stem grooving virus by inserting a termination codon into the variable region located between the putative Rep- and CP-coding regions. These mutants were capable of systemic infection, although their pathogenicity was attenuated. In vitro translation of ORF1 produced both the full-length polyprotein and the smaller Rep protein. The results of in vivo reporter assays suggested that the mechanism of this early termination is a ribosomal -1 frame-shift occurring downstream from the conserved Rep domains. The mechanism of capillovirus gene expression and the very close evolutionary relationship between the genera Capillovirus and Trichovirus are discussed.

Positive selection acting on a surface membrane protein of the plant-pathogenic phytoplasmas.

Phytoplasmas are plant-pathogenic bacteria that cause numerous diseases. This study shows a strong positive selection on the phytoplasma antigenic membrane protein (Amp). The ratio of nonsynonymous to synonymous substitutions was >1 with all the methods we tested. The clear positive selections imply an important biological role for Amp in host-bacterium interactions.

Interaction between the membrane protein of a pathogen and insect microfilament complex determines insect-vector specificity.

Many insect-transmissible pathogens are transmitted by specific insect species and not by others, even if they are closely related. The molecular mechanisms underlying such strict pathogen-insect specificity are poorly understood. Candidatus Phytoplasma asteris, OY strain, line W (OY), is a phytopathogenic bacterium transmitted from plant to plant by sap-feeding insect vectors (leafhoppers). Our study focused on an abundant cell-surface membrane protein of the phytoplasma named antigenic membrane protein (Amp), which is not homologous with any reported functional protein. Immunofluorescence microscopy of the phytoplasma-infected insect showed that OY phytoplasma was localized to the microfilaments of the visceral smooth muscle surrounding the insect's intestinal tract. The affinity column assay showed that Amp forms a complex with three insect proteins: actin, myosin heavy chain, and myosin light chain. Amp-microfilament complexes were detected in all OY-transmitting leafhopper species, but not in the non-OY-transmitting leafhoppers, suggesting that the formation of the Amp-microfilament complex is correlated with the phytoplasma-transmitting capability of leafhoppers. Although several studies have reported interactions between pathogens and mammalian microfilaments, this is an example of host-specific interactions between a bacterial surface protein and a host microfilament in insect cells. Our data also suggest that the utilization of a host microfilament may be a universal system for pathogenic bacteria infecting mammals or insects.

The efficiency of interference of Potato virus X infection depends on the target gene.

RNA silencing is a natural defense response against viral infection. This phenomenon has been used to interfere with viral infections by exploiting fragments of viral genomes as sources of RNA silencing. Agrobacterium-mediated transient expression of a hairpin RNA derived from the TGBp1 gene of Potato virus X (PVX) induced RNA silencing of the TGBp1 gene and resulted in interference of PVX infection. The interference was induced in the infiltrated leaves but not in the upper non-infiltrated leaves. Transient expression of a CP hairpin RNA also induced interference of PVX. The TGBp1 hairpin RNA showed more efficient interference of PVX infection than the CP hairpin RNA.

Phylogenetic characteristics, genomic heterogeneity and symptomatic variation of five closely related Japanese strains of Potato virus X.

To elucidate the genomic determinants of Potato virus X (PVX) strains, which cause diverse responses in host plants, we determined the complete genomic RNA sequences of four Japanese PVX strains: PVX-BS, -BH, -OG, and -TO. These four strains, plus the previously sequenced PVX-OS strain, differ in their pathogenicity in wild potato (Solanum demissum) and tobacco (Nicotiana tabacum cv. Samsun NN). The genomic sequences of these five PVX strains were highly homologous (i.e., the nucleotide sequence identity ranged from 95.4 to 98.5%). Phylogenetic analysis indicated that the Japanese PVX strains originated from an ancestral PVX strain in the European group, and that the virulence of these strains in both S. demissum and tobacco is not correlated with their phylogenetic relationships, suggesting that the pathogenicity of each strain in these host plants is determined by a relatively small number of nucleotides and can easily be altered independent of phylogenetic relationships. Particularly, OS, BH, and BS, which respectively produce markedly contrasting ringspot, mosaic, and asymptomatic infections in tobacco leaves, were the most closely related, suggesting that these three strains are an attractive model for analyzing the genetic determinants causing these symptoms. A possible correlation between the genomic and biological differences of these strains is discussed.

Constitutive expression of human lactoferrin and its N-lobe in rice plants to confer disease resistance.

The milk protein, lactoferrin, is known to have antibacterial, antiviral, and antifungal activities. To explore the possibility of conferring disease resistance in plants by expressing this protein, the gene for the full-length human lactoferrin (HLF), as well as the N-lobe, the N-terminal half molecule (HLFN), was introduced into rice plants and expressed constitutively under the control of the cauliflower mosaic virus 35S promotor. Western blot analysis of leaves from HLF-transgenic rice plants showed an 80 kDa-band, which was about 1-2 kDa less than human milk lactoferrin. HLFN was expressed as a 45-kDa protein and retained its heparin-binding property. Deglycosylation experiments suggested that both proteins produced by the plants had plant-type oligosaccharide chains. The transgenic rice plants were assessed for resistance against disease-causing bacteria, virus, and fungi. Of the pathogens tested, significant resistance against Burkholderia (Pseudomonas) plantarii, the causative agent of bacterial seedling blight disease, was observed in the transgenic plants expressing HLF or HLFN.

A single amino acid residue of RNA-dependent RNA polymerase in the Potato virus X genome determines the symptoms in Nicotiana plants.

A Potato virus X (PVX) strain, PVX-OS, causes a necrotic mosaic in Nicotiana benthamiana and ring spot mosaic in N. tabacum cv. SamsunNN. By contrast, strain PVX-BS causes a mild mosaic in N. benthamiana and systemic asymptomatic infection in N. tabacum cv. SamsunNN. To investigate the viral determinant of this difference, we produced various infectious cDNA clones chimeric between these PVX genomes and clones with point mutations introduced by site-directed mutagenesis. Inoculation tests with these clones mapped the symptom determinant in Nicotiana plants to the 1422 amino acid residue in the region of the C-terminus of RNA-dependent RNA polymerase (RdRp). Western blot analysis and local lesion assay indicated that virus accumulation in the infected leaves was similar for these PVX strains, suggesting that the symptom difference was not due to virus accumulation.

Complementation of sugary-1 phenotype in rice endosperm with the wheat isoamylase1 gene supports a direct role for isoamylase1 in amylopectin biosynthesis.

To examine the role of isoamylase1 (ISA1) in amylopectin biosynthesis in plants, a genomic DNA fragment from Aegilops tauschii was introduced into the ISA1-deficient rice (Oryza sativa) sugary-1 mutant line EM914, in which endosperm starch is completely replaced by phytoglycogen. A. tauschii is the D genome donor of wheat (Triticum aestivum), and the introduced fragment effectively included the gene for ISA1 for wheat (TaISA1) that was encoded on the D genome. In TaISA1-expressing rice endosperm, phytoglycogen synthesis was substantially replaced by starch synthesis, leaving only residual levels of phytoglycogen. The levels of residual phytoglycogen present were inversely proportional to the expression level of the TaISA1 protein, although the level of pullulanase that had been reduced in EM914 was restored to the same level as that in the wild type. Small but significant differences were found in the amylopectin chain-length distribution, gelatinization temperatures, and A-type x-ray diffraction patterns of the starches from lines expressing TaISA1 when compared with wild-type rice starch, although in the first two parameters, the effect was proportional to the expression level of TaISA. The impact of expression levels of ISA1 on starch structure and properties provides support for the view that ISA1 is directly involved in the synthesis of amylopectin.

RERJ1, a jasmonic acid-responsive gene from rice, encodes a basic helix-loop-helix protein.

Differential screening of a cDNA library constructed using poly(A)(+) RNA from suspension-cultured rice cells treated with jasmonic acid (JA) for 1/2h yielded a cDNA of a gene tentatively named RERJ1 that is upregulated in response to exogenous JA. Northern blot analysis indicated that the RERJ1 mRNA levels peaked at 1/2-1h after the addition of jasmonic acid and then decreased gradually. RERJ1 encodes a transcriptional regulator with a basic helix-loop-helix motif. The phenotypes of transgenic rice plants overexpressing sense or antisense RERJ1 mRNA demonstrated that RERJ1 is involved in the growth inhibition of rice shoots caused by JA. Other biological functions of RERJ1 are discussed from an evolutionary standpoint.

Secretion of immunodominant membrane protein from onion yellows phytoplasma through the Sec protein-translocation system in Escherichia coli.

A gene that encodes a putative SecE protein, which is a component of the Sec protein-translocation system, was cloned from the onion yellows phytoplasma (OY). The identification of this gene and the previously reported genes encoding SecA and SecY provides evidence that the Sec system exists in phytoplasma. In addition, a gene encoding an antigenic membrane protein (Amp) (a type of immunodominant membrane protein) of OY was cloned and sequenced. The OY amp gene consisted of 702 nt encoding a protein of 233 aa which was highly similar to Amp of aster yellows phytoplasma (AY). Part of OY Amp was overexpressed in Escherichia coli, purified, and used to raise an anti-Amp polyclonal antibody. The anti-Amp antibody reacted specifically with an OY-infected plant extract in Western blot analysis and was therefore useful for the detection of OY as well as Amp. Amp has a conserved protein motif that is known to be exported by the Sec system of E. coli. A partial OY Amp protein expressed in E. coli was localized in the periplasm as a shorter, putatively processed form of the protein. It had probably been exported from the cytoplasm to the periplasm through the Sec system. Moreover, OY Amp protein expressed in OY and detected in OY-infected plants was apparently also processed. Because phytoplasmas cannot be cultured or transformed, little information is available regarding their protein secretion systems. This study suggests that the Sec system operates in this phytoplasma to export OY Amp.

Reductive evolution suggested from the complete genome sequence of a plant-pathogenic phytoplasma.

The minimal gene set essential for life has long been sought. We report the 860-kb genome of the obligate intracellular plant pathogen phytoplasma (Candidatus Phytoplasma asteris, OY strain). The phytoplasma genome encodes even fewer metabolic functions than do mycoplasma genomes. It lacks the pentose phosphate cycle and, more unexpectedly, ATP-synthase subunits, which are thought to be essential for life. This may be the result of reductive evolution as a consequence of life as an intracellular parasite in a nutrient-rich environment.

An Antibody Against the SecA Membrane Protein of One Phytoplasma Reacts with Those of Phylogenetically Different Phytoplasmas.

ABSTRACT Antisera raised against phloem-limited phytoplasmas generally react only with the phytoplasma strain used to produce the antigen. There is a need for an antiserum that reacts with a variety of phytoplasmas. Here, we show that an antiserum raised against the SecA membrane protein of onion yellows phytoplasma, which belongs to the aster yellows 16S-group, detected eight phytoplasma strains from four distinct 16S-groups (aster yellows, western X, rice yellow dwarf, and elm yellows). In immunoblots, approximately 96-kDa SecA protein was detected in plants infected with each of the eight phytoplasmas. Immunohistochemical staining of thin sections prepared from infected plants was localized in phloem tissues. This antiserum should be useful in the detection and histopathological analysis of a wide range of phytoplasmas.

In planta dynamic analysis of onion yellows phytoplasma using localized inoculation by insect transmission.

ABSTRACT Due to the lack of a means to inoculate plants mechanically, the histological dynamics and in planta spread of phytoplasmas have been studied very little. We analyzed the dynamics of plant infection by phytoplasmas, using a technique to infect a limited area of a leaf, nested polymerase chain reaction (PCR), real-time PCR, and immunohistochemical visualization. Following localized inoculation of a leaf of garland chrysanthemum (Chrysanthemum coronarium) by the vector leafhopper Macrosteles striifrons, the onion yellows (OY) phytoplasma spread within the plant from the inoculated leaf to the main stem (1 day postinoculation [dpi]), to the roots and the top leaf (2 dpi), and to other leaves from top to bottom (from 7 to 21 dpi). The populations of the OY phytoplasmas in inoculated leaves and roots increased approximately sixfold each week from 14 to 28 dpi. At 14 dpi, the OY phytoplasmas colonized limited regions of the phloem tissue in both the root and stem and then spread throughout the phloem by 21 dpi. This information should form the basis for elucidating the mechanisms of phytoplasma multiplication and migration within a plant host.