Battle for control of anthocyanin biosynthesis in two Brassicaceae species infected with turnip mosaic virus.

It has previously been found that turnip mosaic virus (TuMV) greatly suppresses anthocyanin accumulation (AA) in Brassica rapa leaves, and that such leaves become infected whilst anthocyanin-enriched leaves on the same plants are rarely infected. To clarify whether AA is a defense against TuMV, in this study we examined tissue-level patterns of spontaneous AA in relation to the cellular localization of a TuMV strain that expresses a yellow fluorescent protein. We found that TuMV infection was significantly blocked by AA, suggesting that it functions as a chemical barrier against TuMV. We next analysed changes in expression of genes related to anthocyanin biosynthesis in TuMV-infected leaves of Arabidopsis. TuMV also suppressed AA that is induced by high light in Arabidopsis, and this this suppression was mainly due to inhibited expression of anthocyanin late-biosynthesis genes (LBGs). Most positive transcription factors of LBGs were also down-regulated, while the negative regulator SPL15 was highly up-regulated. Cucumber mosaic virus (CMV) also moderately suppressed AA in Arabidopsis, but in a different manner. Since it appeared that anthocyanin-enriched leaves of Arabidopsis were resistant to TuMV but not CMV, our results suggested that the anthocyanin-associated resistance that we observed was specific to TuMV.

Progressive DNA demethylation in epigenetic hybrids between parental plants with and without methylation of the transgene promoter.

KEY MESSAGE: Crosses of parents that differ in their DNA methylation states leads to progressive demethylation in the F1 hybrids. In plant breeding research, hybrid vigor in F1 hybrids is known to be a very important phenomenon. Hybrid vigor, or heterosis, refers to the fact that F1 hybrids from crosses with a certain combination of parents have traits that are superior to those of the parents. In addition, DNA methylation is an important factor that affects gene expression in plant genomes and contributes to hybrid vigor. We introduced the 35S promoter sequence into the cucumber mosaic virus (CMV)-based vector and inoculated the GFP-expressing transgenic Nicotiana benthamiana line 16c with the recombinant virus specifically to induce DNA methylation on the 35S promoter. For plants that had transcriptional gene silencing (TGS) of GFP established by methylation of the 35S promoter (35S-TGS), TGS was fully maintained in their later self-pollinated generations. When the 35S-TGS plants were crossed with 16c, which does not contain DNA methylation in the 35S promoter, the F1 hybrids unexpectedly became progressively DNA demethylated as the plants grew. We hypothesis that in F1 hybrids that are produced by a cross between parents with extremely different gene methylation states, the methylation state of the genes in question may shift more and more to hypomethylation as the plants grow. This progressive demethylation phenomenon observed in this study may be important in plant breeding to reactivate the genes which were silenced by DNA methylation.

Transcriptional silencing of 35S driven-transgene is differentially determined depending on promoter methylation heterogeneity at specific cytosines in both plus- and minus-sense strands.

BACKGROUND: De novo DNA methylation triggered by short interfering RNAs is called RNA-directed DNA methylation (RdDM). Transcriptional gene silencing (TGS) through RdDM can be induced using a viral vector. We have previously induced RdDM on the 35S promoter in the green fluorescent protein (GFP)-expressing Nicotiana benthamiana line 16c using the cucumber mosaic virus vector. The GFP fluorescence phenotype segregated into two types, "red" and "orange" in the first self-fertilized (S1) progeny plants by the difference in degree of recovery from TGS on GFP expression. In the second self-fertilized generation (S2 plants), the phenotypes again segregated. Explaining what generates the red and orange types could answer a very important question in epigenetics: How is the robustness of TGS maintained after RdDM induction? RESULTS: In bisulfite sequencing analyses, we found a significant difference in the overall promoter hypermethylation pattern between the red and orange types in S1 plants but little difference in S2 plants. Therefore, we assumed that methylation at some specific cytosine residues might be important in determining the two phenotypes. To find the factor that discriminates stable, robust TGS from the unstable TGS with incomplete inheritance, we analyzed the direct effect of methylated cytosine residues on TGS. Because it has not yet been demonstrated that DNA methylation at a few specific cytosine residues on known sequence elements can indeed determine TGS robustness, we newly developed a method by which we can directly evaluate the effect of specific methylation on promoter activity. In this assay, we found that the effects of the specific cytosine methylation on TGS differed between the plus- and minus-strands. CONCLUSIONS: We found two distinct phenotypes, the stable and unstable TGS in the progenies of virus-induced TGS plants. Our bisulfite sequencing analyses suggested that methylation at some specific cytosine residues in the 35S promoter played a role in determining whether stable or unstable TGSs are induced. Using the developed method, we inferred that DNA methylation heterogeneity in and between the plus- and minus-strands can differentially determine TGS.

Pid3-I1 is a race-specific partial-resistance allele at the Pid3 blast resistance locus in rice.

KEY MESSAGE: The rice blast resistance QTL detected on chromosome 6 in MC276 is Pid3-I1, one of the multiple alleles at the Pid3 locus. Pid3-I1 shows race-specific partial resistance. Many of the quantitative trait loci (QTLs) for rice blast resistance reported to date remain unidentified. In the present study, we focused on qBRM6.2, a known blast-resistance QTL in experimental resistant rice line MC276. A CO39 near-isogenic line (NIL) carrying qBRM6.2 from MC276 was developed here, and we showed that qBRM6.2 resistance was partial but race specific to Japanese blast isolates using the NIL. Because defense genes in the NIL were expressed sooner than those in CO39 after inoculation with a blast isolate, qBRM6.2 resistance appeared to be an induced resistance. Next, we demonstrated that qBRM6.2 was located within a 123-kb interval on chromosome 6. Among the six genes annotated in the interval, only four genes appeared to be functional. Among these four, a polymorphism between CO39 and the NIL for qBRM6.2 at the amino acid sequence level was detected only in Os06g0330400 that encodes a fatty acid hydroxylase domain-containing protein and in Os06g0330100, the blast resistance locus Pid3, that encodes a nucleotide-binding site-leucine-rich repeat protein. Moreover, the allele at the Pid3 locus in the NIL was Pid3-I1, originally identified as a complete blast resistance gene in Kasalath. To clarify whether Pid3-I1 is qBRM6.2, we investigated the resistance phenotype of Pid3-I1 to Japanese isolates using Nipponbare transgenic lines that express Pid3-I1. The results showed that Pid3-I1 was a race-specific but partial-resistance allele at the Pid3 locus, suggesting strongly that Pid3-I1 is qBRM6.2. The discrepancy in the phenotype of Pid3-I1 between the present and previous reports is also discussed.

Differential Regulation of Starch-synthetic Gene Expression in Endosperm Between Indica and Japonica Rice Cultivars.

BACKGROUND: Grain filling rates (GFRs) of indica rice cultivars are often higher than those of japonica cultivars. Although GFR is mainly determined by the starch accumulation rate (SAR) in endosperm, the genetic basis for SAR during the ripening period has not been well studied in rice. To elucidate the factors influencing the differing SARs between typical indica and japonica cultivars, we focused on differences in sink potentials, especially on starch synthesis in the endosperm. RESULTS: SAR in indica rice cultivar IR36 was significantly higher than in japonica cultivar T65. Although enzymes for both amylose and amylopectin syntheses had higher activity in IR36, amylopectin synthesis was seemingly more important for accelerating SAR because an elevation of amylose synthesis ability alone in the T65 genetic background did not result in the same level of SAR as IR36. In IR36, most starch-synthetic genes (SSGs) in the endosperm were more highly expressed during ripening than in T65. In panicle culture experiments, the SSGs in rice endosperm were regulated in either sucrose-dependent or -independent manners, or both. All SSGs except SSI and BEIIa were responsive to sucrose in both cultivars, and GBSSI, AGPS2b and PUL were more responsive to sucrose in IR36. Interestingly, the GBSSI gene (Wx a ) in IR36 was highly activated by sucrose, but the GBSSI gene (Wx b ) in T65 was insensitive. In sucrose-independent regulation, AGPL2, SSIIIa, BEI, BEIIb and ISA1 genes in IR36 were upregulated 1.5 to 2 times more than those in T65. Additionally, at least SSI and BEIIa might be regulated by unknown signals; that regulation pathway should be more activated in IR36 than T65. CONCLUSIONS: In this study, at least three regulatory pathways seem to be involved in SSG expression in rice endosperm, and all pathways were more active in IR36. One of the factors leading to the high SAR of IR36 seemed to be an increase in the sink potential.

Construction of a System for the Strawberry Nursery Production towards Elimination of Latent Infection of Anthracnose Fungi by a Combination of PCR and Microtube Hybridization.

One of the major problems in strawberry production is difficulty in diagnosis of anthracnose caused by Colletotrichum acutatum or Glomerella cingulata in latent infection stage. We here developed a diagnostic tool for the latent infection consisting of initial culturing of fungi, DNA extraction, synthesis of PCR-amplified probes and microtube hybridization (MTH) using a macroarray. The initial culturing step is convenient to lure the fungi out of the plant tissues, and to extract PCR-inhibitor-free DNA directly from fungal hyphae. For specific detection of the fungi, PCR primers were designed to amplify the fungal MAT1-2 gene. The subsequent MTH step using the PCR products as probes can replace the laborious electrophoresis step providing us sequence information and high-throughput screening. Using this method, we have conducted a survey for a few thousands nursery plants every year for three consecutive years, and finally succeeded in eliminating latent infection in the third year of challenge.

Ascorbic acid accumulates as a defense response to Turnip mosaic virus in resistant Brassica rapa cultivars.

We initially observed that Brassica rapa cultivars containing the Turnip mosaic virus (TuMV) resistance gene, Rnt1-1, accumulated a high level of endogenous ascorbic acid (AS) and dehydroascobic acid (DHA) when infected with TuMV. We here hypothesized a possible contribution of an elevated level of AS+DHA (TAA) to the Rnt1-1-mediated resistance, and conducted a series of experiments using B. rapa and Arabidopsis plants. The application of l-galactose (the key substrate in AS synthesis) to a susceptible cultivar could increase the TAA level ~2-fold, and simultaneously lead to some degree of enhanced viral resistance. To confirm some positive correlation between TAA levels and viral resistance, we analyzed two Arabidopsis knockout mutants (ao and vtc1) in the AS pathways; the TAA levels were significantly increased and decreased in ao and vtc1 plants, respectively. While the ao plants showed enhanced resistance to TuMV, vtc1 plants were more susceptible than the control, supporting our hypothesis. When we analyzed the expression profiles of the genes involved in the AS pathways upon TuMV infection, we found that the observed TAA increase was mainly brought about by the reduction of AS oxidation and activation of AS recycling. We then investigated the secondary signals that regulate endogenous TAA levels in response to viral infection, and found that jasmonic acid (JA) might play an important role in TAA accumulation. In conclusion, we reason that the elevated TAA accumulation in B. rapa plants would be at least partly mediated by the JA-dependent signaling pathway and may significantly contribute to viral resistance.

Chemical Promotion of Endogenous Amounts of ABA in Arabidopsis thaliana by a Natural Product, Theobroxide.

Plant hormones are a group of structurally diverse small compounds that orchestrate the cellular processes governing proper plant growth and environmental adaptation. To understand the details of hormonal activity, we must study not only their inherent activities but also the cross-talk among plant hormones. In addition to their use in agriculture, plant chemical activators, such as probenazole and uniconazole, have made great contributions to understand hormonal cross-talk. However, the use of plant chemical activators is limited due to the lack of activators for certain hormones. For example, to the best of our knowledge, there are only a few chemical activators previously known to stimulate the accumulation of ABA in plants, such as absinazoles and proanthocyanidins. In many cases, antagonistic effects have been examined in experiments using exogenously applied ABA, although these studies did not account for biologically relevant concentrations. In this report, it was found that a natural product, theobroxide, had potential as a plant chemical activator for stimulating the accumulation of ABA. Using theobroxide, the antagonistic effect of ABA against GAs was proved without exogenously applying ABA or using mutant plants. Our results suggest that ABA levels could be chemically controlled to elicit ABA-dependent biological phenomena.

Combinations of a host resistance gene and the CI gene of turnip mosaic virus differentially regulate symptom expression in Brassica rapa cultivars.

In the pathosystem of Brassica rapa and turnip mosaic virus (TuMV), the type of symptoms expressed by susceptible plants are determined by the gene combinations between the host cultivar and virus strain. In this study, we found that the resistance reaction and symptoms such as systemic lethal necrosis, leaf malformation and mosaic were differentially determined, depending on the combinations of the genotypes for a host locus or two closely linked host loci and the viral CI gene. Systemic necrosis caused by TuMV-UK1 on some B. rapa subsp. pekinensis cultivars is induced in conjunction with a recessive gene, rnt1-2 (resistance and necrosis to tumv 1-2), which is allelic or closely linked to TuMV resistance gene Rnt1-1 on chromosome R6. rnt1-2 is incompletely recessive to rnt1-3, which does not cause any necrotic responses. The genotype rnt1-2/rnt1-3 caused a mild necrosis along leaf veins of severely malformed leaves. A spontaneous mutant, TuMV-UK1 (UK1m), with the amino acid substitution V1827E in CI, broke Rnt1-1 resistance and altered the systemic necrosis and leaf malformation induced by rnt1-2. This single amino acid in the CI protein of UK1 was also associated with severe mosaic and abnormal leaf development, perhaps interacting with unknown host factors. To clarify the relationship between Rnt1-1 and TuRB01b, which was previously reported as a TuMV-UK1 resistance gene on chromosome R6, the B. rapa cultivar Tropical Delight carrying TuRB01b was inoculated with UK1m or the infectious UK1 clone with the CI V1827E mutation. Because Tropical Delight showed resistance to both mutants, Rnt1-1 might be different from TuRB01b.

Multigenic system controlling viral systemic infection determined by the interactions between Cucumber mosaic virus genes and quantitative trait loci of soybean cultivars.

Soybean 'Harosoy' is resistant to Cucumber mosaic virus soybean strain C (CMV-SC) and susceptible to CMV-S strain D (CMV-SD). Using enzyme-linked immunosorbent assay and Northern hybridization, we characterized the Harosoy resistance and found that CMV-SC did not spread systemically but was restricted to the inoculated leaves in Harosoy. Harosoy resistance was not controlled by either a dominant or recessive single gene. To dissect this system controlling long-distance movement of CMV in soybean, we constructed infectious cDNA clones of CMV-SC and CMV-SD. Using these constructs and the chimeric RNAs, we demonstrated that two viral components were required for systemic infection by the virus. The region including the entire 2b gene and the 5' region of RNA3 (mainly the 5' untranslated region) together were required. By quantitative trait locus (QTL) analysis using an F(2) population and the F(3) families derived from Harosoy and susceptible 'Nemashirazu', we also showed that at least three QTLs affected systemic infection of CMV in soybean. Our study on Harosoy resistance to CMV-SC revealed an interesting mechanism, in which multiple host and viral genes coordinately controlled viral systemic infection.

The P3 protein of turnip mosaic virus can alone induce hypersensitive response-like cell death in Arabidopsis thaliana carrying TuNI.

Strains TuR1 and TuC of Turnip mosaic virus (TuMV) induce different symptoms on Arabidopsis thaliana ecotype Landsberg erecta (Ler); plants infected with TuR1 develop systemic necrosis, while TuC causes mosaics. We previously found that the Ler systemic necrosis was controlled by a single dominant gene, TuNI (TuMV necrosis inducer), and that it was actually a form of host defense response leading to a hypersensitive reaction (HR)-like cell death. To identify the viral factor interacting with TuNI, the domain swapping between the genomic clones of TuR1 and TuC was carried out, and we identified the TuMV symptom determinant interacting with TuNI as the P3 gene. Moreover, it was found that the central 0.5-kb domain of P3, including three different amino acids between the two isolates, was responsible for the systemic HR. To verify that the P3 gene can alone induce necrosis, we analyzed the constitutive P3 expression in Ler transgenic plants and the transient P3 expression in Ler protoplasts. These results indicated that P3 alone caused HR-like cell death. In this study, we successfully demonstrated that the systemic necrosis by TuMV in Arabidopsis was determined by the gene-for-gene interaction between TuNI and P3 using the protoplast system for direct verification.

Veinal necrosis induced by turnip mosaic virus infection in Arabidopsis is a form of defense response accompanying HR-like cell death.

In the pathosystems of Turnip mosaic virus (TuMV) with Brassicaceae crops, various symptoms, including mosaic and necrosis, are observed. We previously reported a necrosis-inducing factor TuNI in Arabidopsis thaliana, a model species. In this study, we show that the necrotic symptom induced by TuNI, observed along the veins, was actually a form of defense response accompanying a hypersensitive reaction (HR)-like cell death in the veinal area. The virus is often localized in the necrotic region. The necrotic response is associated with the production of H2O2, accumulation of salicylic acid (SA), emission of ethylene, and subsequent expression of defense-related genes. Additionally, this HR-like cell death is eased or erased by a shading treatment. These features are similar to the HR-associated resistance reaction to pathogens. However, unlike HR, two phytohormones--SA and ethylene--are involved in the necrosis induction, and both SA- and ethylene-dependent pathogenesis-related genes are activated. We concluded that the veinal necrosis induced by TuMV is regulated by a complex and unique network of at least two signaling pathways, which differs from the signal transduction for the known HR-associated resistance.

RMo1 confers blast resistance in barley and is located within the complex of resistance genes containing Mla, a powdery mildew resistance gene.

Isolates of Magnaporthe oryzae (the causal agent of rice blast disease) can infect a range of grass species, including barley. We report that barley Hordeum vulgare cv. Baronesse and an experimental line, BCD47, show a range of resistance reactions to infection with two rice blast isolates. The complete resistance of Baronesse to the isolate Ken 54-20 is controlled by a single dominant gene, designated RMo1. RMo1 mapped to the same linkage map position on chromosome 1H as the powdery mildew resistance locus Mla and an expressed sequence tag (k04320) that corresponds to the barley gene 711N16.16. A resistance quantitative trait locus (QTL), at which Baronesse contributed the resistance allele, to the isolate Ken 53-33 also mapped at the same position as RMo1. Synteny analysis revealed that a corresponding region on rice chromosome 5 includes the bacterial blight resistance gene xa5. These results indicate that a defined region on the short arm of barley chromosome 1H, including RMo1 and Mla, harbors genes conferring qualitative and quantitative resistance to multiple pathogens. The partial resistance of BCD47 to Ken53-33 is determined by alleles at three QTL, two of which coincide with the linkage map positions of the mildew resistance genes mlo and Mlf.

Fine genetic mapping of the TuNI locus causing systemic veinal necrosis by turnip mosaic virus infection in Arabidopsis thaliana.

In the pathosystem of turnip mosaic virus (TuMV) and Arabidopsis thaliana, two distinct symptoms (mosaic symptom and veinal necrosis) were observed that were dependent upon the combination of the TuMV isolate and the Arabidopsis ecotype. The Col-0 ecotype developed mosaic symptoms after infection with the TuMV isolate Azu while the Ler ecotype developed veinal necrosis after infection with the same TuMV isolate. The Ler phenotype is controlled by a single dominant gene TuNI (TuMV necrosis inducer) which is located on chromosome 1. The TuNI gene was precisely mapped to the ~105 kb interval between the two markers of mXF41 and mRF28 by using several types of DNA polymorphism markers. Within this region, which included largely duplicated sequences, a total of 19 putative genes were predicted and 15 of these were classified into five gene families. The genes belonging to the gene families At1g58480 and At1g58602 may function in response to infection by pathogens. The gene family At1g58480 encodes lipase-like proteins, which might be involved in the induction of defence responses that are mediated by salicylic acid. The gene family At1g58602 encodes the CC-NBS-LRR (CNL) proteins, which are known to function as one of the plant resistance (R) proteins against pathogens. In the present study, the possibility that TuNI might function as an R gene was discussed.

Sequence rearrangement in the AT-rich minisatellite of the novel rice transposable element Basho.

In the process of characterizing a rice wx deletion mutant, an AT-rich minisatellite sequence that consisted of units of approximately 80 bp was detected about 2.3 kb downstream of the wx gene. This AT-rich minisatellite was a multiple-copy element (1 x 10(3) to 2 x 10(3) copies per haploid genome) and interspersed in the rice genome. By BLAST homology search it was indicated that not only the tandem repeat but also both flanking sequences were conserved among copies. According to the characteristics of the termini (5'-CHH ... CTAG-3') and a target site preference for T, this AT-rich minisatellite accompanying the flanking sequences was classified into a novel transposon, Basho. The results of direct amplification of Basho showed that relatively large variation in size existed in the Basho family. We estimate the variation to be generated by not only alteration of the number of units in the minisatellite but also by duplications of larger blocks including the conserved flanking sequences caused by single-strand mispairing (SSM) at noncontiguous repeats. Because the AT-rich minisatellite contained in Basho possessed several motifs of the matrix attachment region (MAR) in its repeat unit, the functional role as MAR in the rice genome was discussed.