Conferring virus resistance in tomato by independent RNA silencing of three tomato homologs of Arabidopsis TOM1.

The TOM1/TOM3 genes from Arabidopsis are involved in the replication of tobamoviruses. Tomato homologs of these genes, LeTH1, LeTH2 and LeTH3, are known. In this study, we examined transgenic tomato lines where inverted repeats of either LeTH1, LeTH2 or LeTH3 were introduced by Agrobacterium. Endogenous mRNA expression for each gene was detected in non-transgenic control plants, whereas a very low level of each of the three genes was found in the corresponding line. Small interfering RNA was detected in the transgenic lines. Each silenced line showed similar levels of tobamovirus resistance, indicating that each gene is similarly involved in virus replication.

Conferring high-temperature tolerance to nontransgenic tomato scions using graft transmission of RNA silencing of the fatty acid desaturase gene.

We investigated graft transmission of high-temperature tolerance in tomato scions to nontransgenic scions from transgenic rootstocks, where the fatty acid desaturase gene (LeFAD7) was RNA-silenced. Tomato was transformed with a plasmid carrying an inverted repeat of LeFAD7 by Agrobacterium. Several transgenic lines showed the lower amounts of LeFAD7 RNA and unsaturated fatty acids, while nontransgenic control did not, and siRNA was detected in the transgenic lines, but not in control. These lines grew under conditions of high temperature, while nontransgenic control did not. Further, the nontransgenic plants were grafted onto the silenced transgenic plants. The scions showed less of the target gene RNA, and siRNA was detected. Under high-temperature conditions, these grafted plants grew, while control grafted plants did not. Thus, it was shown that high-temperature tolerance was conferred in the nontransgenic scions after grafting onto the silenced rootstocks.

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.

Overexpression of a rice heme activator protein gene (OsHAP2E) confers resistance to pathogens, salinity and drought, and increases photosynthesis and tiller number.

Heme activator protein (HAP), also known as nuclear factor Y or CCAAT binding factor (HAP/NF-Y/CBF), has important functions in regulating plant growth, development and stress responses. The expression of rice HAP gene (OsHAP2E) was induced by probenazole (PBZ), a chemical inducer of disease resistance. To characterize the gene, the chimeric gene (OsHAP2E::GUS) engineered to carry the structural gene encoding ß-glucuronidase (GUS) driven by the promoter from OsHAP2E was introduced into rice. The transgenic lines of OsHAP2Ein::GUS with the intron showed high GUS activity in the wounds and surrounding tissues. When treated by salicylic acid (SA), isonicotinic acid (INA), abscisic acid (ABA) and hydrogen peroxide (H2 O2 ), the lines showed GUS activity exclusively in vascular tissues and mesophyll cells. This activity was enhanced after inoculation with Magnaporthe oryzae or Xanthomonas oryzae pv. oryzae. The OsHAP2E expression level was also induced after inoculation of rice with M. oryzae and X. oryzae pv. oryzae and after treatment with SA, INA, ABA and H2 O2, respectively. We further produced transgenic rice overexpressing OsHAP2E. These lines conferred resistance to M. oryzae or X. oryzae pv. oryzae and to salinity and drought. Furthermore, they showed a higher photosynthetic rate and an increased number of tillers. Microarray analysis showed up-regulation of defence-related genes. These results suggest that this gene could contribute to conferring biotic and abiotic resistances and increasing photosynthesis and tiller numbers.

Functional differentiation in the leucine-rich repeat domains of closely related plant virus-resistance proteins that recognize common avr proteins.

The N' gene of Nicotiana sylvestris and L genes of Capsicum plants confer the resistance response accompanying the hypersensitive response (HR) elicited by tobamovirus coat proteins (CP) but with different viral specificities. Here, we report the identification of the N' gene. We amplified and cloned an N' candidate using polymerase chain reaction primers designed from L gene sequences. The N' candidate gene was a single 4143 base pairs fragment encoding a coiled-coil nucleotide-binding leucine-rich repeat (LRR)-type resistance protein of 1,380 amino acids. The candidate gene induced the HR in response to the coexpression of tobamovirus CP with the identical specificity as reported for N'. Analysis of N'-containing and tobamovirus-susceptible N. tabacum accessions supported the hypothesis that the candidate is the N' gene itself. Chimera analysis between N' and L(3) revealed that their LRR domains determine the spectrum of their tobamovirus CP recognition. Deletion and mutation analyses of N' and L(3) revealed that the conserved sequences in their C-terminal regions have important roles but contribute differentially to the recognition of common avirulence proteins. The results collectively suggest that Nicotiana N' and Capsicum L genes, which most likely evolved from a common ancestor, differentiated in their recognition specificity through changes in the structural requirements for LRR function.

Molecular analysis of transgenic melon plants showing virus resistance conferred by direct repeat of movement gene of Cucumber green mottle mosaic virus.

Cucumber green mottle mosaic virus (CGMMV) is a major limiting factor in the production of melon plants worldwide. For effective control of this virus using the transgenic approach, the direct repeat of the movement protein gene of CGMMV was used for transforming melon plants by Agrobacterium tumefaciens. PCR and Southern blot analyses of T3 confirmed that they carried the transgene. Northern blot analysis with total RNA showed that transgene transcript RNA as well as siRNA was observed in all plants tested. Separate leaves or individual plants were inoculated with CGMMV and subjected to ELISA and RNA blot analysis using the coat protein gene probe of the virus. Compared to nontransgenic control, these plants were shown to have high virus resistance. Furthermore, cytosine of the transgene DNA in the plants was methylated. Thus, these results reveal that the transgenic lines were highly resistant to the virus through RNA silencing. Key message High virus resistance was obtained in transgenic melon plants with direct repeat of movement protein gene of Cucumber green mottle mosaic tobamovirus through RNA silencing.

Cloning and expression of the isocitrate lyase gene from a nitrogen-fixing bacterium, Azotobacter vinelandii, and functional analysis of the enzyme by site-directed mutagenesis.

The gene encoding isocitrate lyase (ICL) from a nitrogen-fixing mesophilic bacterium, Azotobacter vinelandii strain IAM1078, was cloned, and the gene expression was examined. When sodium acetate or glucose was used as carbon source, similar growth was observed in this bacterium, but the ICL activity of cells grown with the former source was 43-hold higher than those with the latter. In addition, northern blot analysis revealed that expression of the ICL gene was induced by acetate. Based on a comparison of the amino acid sequences of the ICLs of various organisms, the ICL of this bacterium was found to be classifiable into subfamily 3, one of two phylogenetic groups of eubacteial ICLs. Replacement of the Ile504 in the ICL by Met, which is conserved in the corresponding position of cold-adapted ICLs of psychrophlic bacteria, resulted in decreased thermostability of activity, indicating that this amino acid residue is involved in thermal properties of this enzyme.

RACE1, a Japanese Blumeria graminis f. sp. hordei isolate, is capable of overcoming partially mlo-mediated penetration resistance in barley in an allele-specific manner.

Loss-of-function mutation of the MILDEW RESISTANCE LOCUS O (Mlo) gene confers durable and broad-spectrum resistance to powdery mildew fungi in various plants, including barley. In combination with the intracellular nucleotide-binding domain and leucine-rich repeat receptor (NLR) genes, which confer the race-specific resistance, the mlo alleles have long been used in barley breeding as genetic resources that confer robust non-race-specific resistance. However, a Japanese Blumeria graminis f. sp. hordei isolate, RACE1, has been reported to have the potential to overcome partially the mlo-mediated penetration resistance, although this is yet uncertain because the putative effects of NLR genes in the tested accessions have not been ruled out. In this study, we examined the reproducibility of the earlier report and found that the infectious ability of RACE1, which partially overcomes the mlo-mediated resistance, is only exerted in the absence of NLR genes recognizing RACE1. Furthermore, using the transient-induced gene silencing technique, we demonstrated that RACE1 can partially overcome the resistance in the host cells with suppressed MLO expression but not in plants possessing the null mutant allele mlo-5.

Biological properties of flutianil as a novel fungicide against powdery mildew.

Flutianil, chemically characterized as a cyano-methylene thiazolidine, showed antifungal activity against powdery mildew on various crops but not any other plant pathogens tested. Flutianil showed high residual and translaminar activities and rainfastness against Podosphaera xanthii. It also exhibited curative activity against P. xanthii on cucumber at an extremely low concentration of 10 mg/L. There was no cross-resistance between flutianil and other existing fungicides. Morphological studies revealed that flutianil did not inhibit the early infection behavior of Blumeria graminis f. sp. hordei i.e., conidium bursting, primary and appressorial germinations, appressorium development and hook formations, but it did inhibit haustorium formation and further fungal development. Nutrient absorption by haustoria and subsequent secondary hyphal elongation was inhibited by flutianil but not by the fungicide that showed a similar inhibitory pattern up to the haustorium formation stage of the infection process. These findings suggest that flutianil might have a novel mode of action.

Fast and Inexpensive Phenotyping and Genotyping Methods for Evaluation of Barley Mutant Population.

To further develop barley breeding and genetics, more information on gene functions based on the analysis of the mutants of each gene is needed. However, the mutant resources are not as well developed as the model plants, such as Arabidopsis and rice. Although genome editing techniques have been able to generate mutants, it is not yet an effective method as it can only be used to transform a limited number of cultivars. Here, we developed a mutant population using 'Mannenboshi', which produces good quality grains with high yields but is susceptible to disease, to establish a Targeting Induced Local Lesions IN Genomes (TILLING) system that can isolate mutants in a high-throughput manner. To evaluate the availability of the prepared 8043 M3 lines, we investigated the frequency of mutant occurrence using a rapid, visually detectable waxy phenotype as an indicator. Four mutants were isolated and single nucleotide polymorphisms (SNPs) were identified in the Waxy gene as novel alleles. It was confirmed that the mutations could be easily detected using the mismatch endonuclease CELI, revealing that a sufficient number of mutants could be rapidly isolated from our TILLING population.

High Humidity Causes Abnormalities in the Process of Appressorial Formation of Blumeria graminis f. sp. hordei.

High humidity decreases the penetration rate of barley powdery mildew Blumeria graminis f. sp. hordei. However, the mechanism is not well understood. In this study, the morphological and cytochemical analyses revealed that substances containing proteins leaked from the tip of the appressorial germ tube of conidia without the formation of appressorium under a high humidity condition. In addition, exposure to high humidity prior to the formation of appressorium caused the aberrant formation of the appressorial germ tube without appressorium formation, resulting in failure to penetrate the host cell. These findings suggest that the formation and maturation of the appressorium requires a low humidity condition, and will be clues to improve the disease management by humidity control.

OsRecQ1, a QDE-3 homologue in rice, is required for RNA silencing induced by particle bombardment for inverted repeat DNA, but not for double-stranded RNA.

Based on the nucleotide sequence of QDE-3 in Neurospora crassa, which is involved in RNA silencing, rice (Oryza sativa) mutant lines disrupted by the insertion of the rice retrotransposon Tos17 were selected. Homozygous individuals from the M(1) and M(2) generations were screened and used for further analyses. The expression of the gene was not detected in leaves or calli of the mutant lines, in contrast to the wild type (WT). Induction of RNA silencing by particle bombardment was performed to investigate any effects of the OsRecQ1 gene on RNA silencing with silencing inducers of the GFP (green fluorescence protein)/GUS (beta-glucuronidase) gene in the mutant lines. The results showed that OsRecQ1 is required for RNA silencing induced by particle bombardment for inverted-repeat DNA, but not for double-stranded RNA (dsRNA). The levels of transcripts from inverted-repeat DNA were much lower in the mutant lines than those in the WT. Furthermore, no effects were observed in the accumulation of endogenous microRNAs (miR171 and miR156) and the production of the short interspersed nuclear element retroelement by small interfering RNA. On the basis of these results, we propose that OsRecQ1 may participate in the process that allows inverted repeat DNA to be transcribed into dsRNA, which can trigger RNA silencing.

Cytosine methylation is associated with RNA silencing in silenced plants but not with systemic and transitive RNA silencing through grafting.

RNA silencing is often associated with methylation of the target gene. The DNA methylation level of transgenes was investigated in post-transcriptionally silenced or non-silenced Nicotiana benthamiana carrying either the 5' region (200 or 400 bp) or the entire region of the coat protein gene (CP, including the 3' non-translated region) of Sweet potato feathery mottle virus. Higher levels of transgene cytosine methylation were observed in both symmetrical (CpG, CpNpG) and non-symmetrical (CpHpH) contexts (CpG>CpNpG>CpHpH) in silenced lines, but there was very lower levels or no transgene methylation in non-silenced lines. RNA silencing was induced in non-silenced scions from silenced rootstocks and spread to the 3' region of the transgene mRNA (Haque et al., Plant Mol. Biol. 2007; 63: 35-47). In this system, transgene methylation levels were analyzed in scions at different time intervals after being grafted onto silenced or non-silenced rootstocks to investigate if transgene methylation was associated with induction or transitivity of RNA silencing. We observed that, there was no change of transgene methylation level in the initial target or in extended regions in scions. These results showed that transgene methylation was associated with RNA silencing in individual transformants, but it was not associated with systemic RNA silencing and/or transitive RNA silencing through grafting.

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.

Response of an aspartic protease gene OsAP77 to fungal, bacterial and viral infections in rice.

BACKGROUND: Aspartic protease (APs) plays important roles in plant growth, development and biotic and abiotic stresses. We previously reported that the expression of a rice AP gene (OsAP77, Os10g0537800) was induced by probenazole (PBZ), a chemical inducer of disease resistance. In this study we examined some characteristics of this gene in response to fungal, bacterial and viral pathogens. RESULTS: To elucidate the spatial and temporal expression of OsAP77, the chimeric gene was constructed carrying the structural gene encoding ß-glucuronidase (GUS) driven by the OsAP77 promoter. This construct was introduced into rice and the transgenic lines were tested to analyze gene expression by fungal, bacterial and viral infections. Inoculation with Magnaporthe oryzae or Xanthomonas oryzae pv. oryzae revealed the enhanced GUS activities in vascular tissues surrounding the symptom sites by each pathogen. Moreover, GUS activity also increased after inoculation with Cucumber mosaic virus (CMV). Transgenic plants immersed in a solution containing salicylic acid (SA), isonicotinic acid (INA), hydrogen peroxide (H2O2) or abscisic acid (ABA) showed an increased level of GUS activity exclusively in vascular tissues. RT-PCR analysis showed that OsAP77 was induced not only by infection with these pathogens, but also after treatment with SA, INA, H2O2 or ABA. A knockout mutant line of OsAP77 by the insertion of Tos17 after inoculation with M. oryzae, X. oryzae pv. oryzae or CMV showed an enhanced susceptibility compared to wild type. CONCLUSION: These results suggest that the expression of OsAP77 is induced by pathogen infection and defense related signaling molecules in a vascular tissue specific manner and that this gene has a positive role of defense response against fungal, bacterial and viral infections.

Both OsRecQ1 and OsRDR1 are required for the production of small RNA in response to DNA-damage in rice.

Small RNA-mediated gene silencing pathways play important roles in the regulation of development, genome stability and various stress responses in many eukaryotes. Recently, a new type of small interfering RNAs (qiRNAs) approximately 20-21 nucleotides long in Neurospora crassa have been shown to mediate gene silencing in the DNA damage response (DDR) pathway. However, the mechanism for RNA silencing in the DDR pathway is largely unknown in plants. Here, we report that a class of small RNAs (qiRNAs) derived from rDNA was markedly induced after treatment by DNA-damaging agents [ethyl methanesulphonate (EMS and UV-C)], and that aberrant RNAs (aRNAs) as precursors were also highly induced after the DNA damage treatment in rice. However, these RNAs were completely abolished in OsRecQ1 (RecQ DNA helicase homologue) and OsRDR1 (RNA-dependent RNA polymerase homologue) mutant lines where either gene was disrupted by the insertion of rice retrotransposon Tos17 after the same treatment. DNA damage resulted in a more significant increase in cell death and a more severe inhibition of root growth in both mutant lines than in the WT. Together, these results strongly suggest that both OsRecQ1 and OsRDR1 play a pivotal role in the aRNA and qiRNA biogenesis required for the DDR and repair pathway in rice, and it may be a novel mechanism of regulation to the DDR through the production of qiRNA in plants.

Graft transmission of RNA silencing to non-transgenic scions for conferring virus resistance in tobacco.

RNA silencing is a mechanism of gene regulation by sequence specific RNA degradation and is involved in controlling endogenous gene expression and defense against invasive nucleic acids such as viruses. RNA silencing has been proven to be transmitted between scions and rootstocks through grafting, mostly using transgenic plants. It has been reported that RNA silencing of tobacco endogenous genes, NtTOM1 and NtTOM3, that are required for tobamovirus multiplication, resulted in high resistance against several tobamoviruses. In the present study, we examined the graft transmission of RNA silencing for conferring virus resistance to non-transgenic scions of the same and different Nicotiana species grafted onto rootstocks in which both NtTOM1 and NtTOM3 were silenced. Non-transgenic Nicotiana tabacum (cvs. Samsun and Xanthi nc) and N. benthamiana were used as scions for grafting onto the rootstocks silenced with both genes. Short interfering RNA (siRNA) of NtTOM1 and NtTOM3 was detected in both the scions and the rootstocks eight weeks after grafting. The leaves were detached from the scions and inoculated with several tobamoviruses. The virus accumulation was tested by ELISA and northern blot analysis. The viruses were detected in grafted scions at extremely low levels, showing that virus resistance was conferred. These results suggest that RNA silencing was induced in and virus resistance was conferred to the non-transgenic scions by grafting onto silenced rootstocks. The effect of low temperature on siRNA accumulation and virus resistance was not significantly observed in the scions.

Gene cloning of cold-adapted isocitrate lyase from a psychrophilic bacterium, Colwellia psychrerythraea, and analysis of amino acid residues involved in cold adaptation of this enzyme.

The gene (icl) encoding cold-adapted isocitrate lyase (ICL) of a psychrophilic bacterium, Colwellia psychrerythraea, was cloned and sequenced. Open reading frame of the gene was 1,587 bp in length and corresponded to a polypeptide composed of 528 amino acids. The deduced amino acid sequence showed high homology with that of cold-adapted ICL from other psychrophilic bacterium, C. maris (88% identity), but the sequential homology with that of the Escherichia coli ICL was low (28% identity). Primer extension analysis revealed that transcriptional start site for the C. psychrerythraea icl gene was guanine, located at 87 bases upstream of translational initiation codon. The expression of this gene in the cells of an E. coli mutant defective in ICL was induced by not only low temperature but also acetate. However, cis-acting elements for cold-inducible expression known in the several other bacterial genes were absent in the promoter region of the C. psychrerythraea icl gene. The substitution of Ala214 for Ser in the C. psychrerythraea ICL introduced by point mutation resulted in the increased thermostability and lowering of the specific activity at low temperature, indicating that Ala214 is important for psychrophilic properties of this enzyme.

The cold-inducible icl gene encoding thermolabile isocitrate lyase of a psychrophilic bacterium, Colwellia maris.

The gene encoding isocitrate lyase (ICL; EC 4.1.3.1) of a psychrophilic bacterium, Colwellia maris, was cloned and sequenced. The ORF of the gene (icl) was 1584 bp long, and the predicted gene product consisted of 528 aa (molecular mass 58150 Da) and showed low homology with the corresponding enzymes from other organisms. The analyses of amino acid content and primary structure of the C. maris ICL suggested that it possessed many features of a cold-adapted enzyme. Primer extension and Northern blot analyses revealed that two species of the icl mRNAs with differential lengths of 5'-untranslated regions (TS1 and TS2) were present, of which the 5' end (TS1 and TS2 sites) were G and A, located at 130 and 39 bases upstream of the translation start codon, respectively. The levels of TS1 and TS2 mRNAs were increased by both acetate and low temperature. The induction of icl expression by low temperature took place in the C. maris cells grown on succinate as the carbon source but not acetate. Furthermore, a similar manner of inductions was also found in the levels of the translation and the enzyme activity in cell-free extract. These results suggest that the icl gene, encoding thermolabile isocitrate lyase, of C. maris is important for acetate utilization and cold adaptation.