Identification and Fine Mapping of Gummy Stem Blight Resistance Gene Gsb-7(t) in Melon.

Gummy stem blight (GSB), caused by Didymella bryoniae, is a devastating fungal disease of melon worldwide. Breeding GSB-resistant cultivars with host resistance genes is considered the most economic and effective strategy to control this disease. In this study, 260 melon germplasm resources were screened for resistance to GSB, and an inbred line, H55R, that exhibited immunity to GSB was identified. To further understand the resistance mechanism of H55R against GSB, an F2 population was obtained from a cross between the GSB-susceptible line A15 and H55R, and genetic analysis indicated that the GSB resistance in H55R was controlled by a single dominant gene, tentatively named Gsb-7(t). The Gsb-7(t) gene was finally delimited to a 140-kb interval on chromosome 7 using bulked segregant analysis and chromosome walking strategies. Ten putative genes were annotated in this region that contains a wall-associated receptor kinase (WAK) gene MELO3C010403. The MELO3C010403 gene contains two alternative transcripts, MELO3C010403-T1 and MELO3C010403-T2, with five and seven nonsynonymous mutation sites, respectively. Gene expression analysis showed that expression of MELO3C010403-T2 but not MELO3C010403-T1 was significantly induced by D. bryoniae at 24 h postinoculation, indicating that the MELO3C010403-T2 transcript of MELO3C010403 was the most likely candidate gene of Gsb-7(t). Our results offer new genetic resources and will be helpful for the development of GSB-resistant melon cultivars in the future.

First Report of Broad Bean Wilt Virus 2 Infecting Balsam (Impatiens balsamina) in China.

Balsam (Impatiens balsamina L.) is an ornamental plant cultivated extensively in China and elsewhere, but it has also been used as a medicinal plant for thousands of years (Qian et al., 2023). In 2022, an examination of 10 garden-grown I. balsamina plants in Chaoyang, Beijing, China revealed eight plants with blotches, mosaic symptoms, and deformed leaves (Fig. S1A). Total RNA was extracted from the symptomatic leaf tissue of these eight plants using the TRIzol reagent (Invitrogen, USA). Four RNA preparations (high quality and quantity) were combined for the small RNA sequencing analysis (TIANGEN Biotech Co., China). A total of 16,509,586 clean reads (18-30 nt) were obtained and assembled into larger contigs using Velvet 1.0.5. A search of the National Center for Biotechnology Information non-redundant database using BLASTX indicated 72, 24, and 19 contigs were homologous to broad bean wilt virus 2 (BBWV2), cucumber mosaic virus (CMV), and impatiens cryptic virus 1 (ICV1) sequences (Zheng et al., 2022), respectively. To verify the next-generation sequencing data, the following three sets of primer pairs were designed according to the contig sequences of these three viruses: CMV-F:5'-ATGGACAAATCTGAATCAACCAGTGC-3'/CMV-R: 5'-CCGTAAGCTGGATGGACAACC-3'; BBWV2-F:5'-CAATTTGGACAACTACAATTTGCC-3'/ BBWV2-R: 5'-GCTGAGTCTAAATCCCATCTATC-3'; and ICV1-F: 5'-CGCACAACT CTACAAT GACATGGTC-3'/ICV1-R: 5'-AGTTCCATCGTCCAGTAGGCG-3'. The primers were used to amplify CMV, BBWV2, and ICV1 sequences by reverse transcription-polymerase chain reaction (RT-PCR), with individual RNA preparations serving as the template. The CMV, BBWV2, and ICV1 target sequences were amplified from eight, four, and four samples, respectively (Fig. S1B). To evaluate virus infectivity, Nicotiana benthamiana seedlings were inoculated using a leaf tissue extract prepared from an infected I. balsamina plant. At 7 days post-inoculation, disease symptoms were detected on N. benthamiana systemic leaves (e.g., deformation and apical necrosis) (Fig. S1C). Confirmation tests involving RT-PCR indicated the N. benthamiana plants were infected with BBWV2 and CMV, but not with ICV1 (Fig. S1D). To obtain the complete BBWV2 genome sequence (RNA1 and RNA2), virus-specific PCR primers (Table S1) were designed to produce the terminal sequences via 5' and 3' rapid amplification of cDNA ends (RACE), which was completed using the SMARTer RACE 5'/3' Kit (Clontech, China). The RNA1 and RNA2 sequences comprised 5,957 nt (GenBank: OQ857921) and 3,614 nt (GenBank: OQ857922), respectively. The BLAST analyses revealed RNA1 and RNA2 were similar to sequences in other BBWV2 isolates (sequence identities of 78.88% to 95.15% and 80.83% to 91.51%, respectively). Using the neighbor-joining method and MEGA 7.0, the phylogenetic relationships between the BBWV2 isolated in this study and other isolates were determined on the basis of the full-length RNA1 and RNA2 sequences (Kumar et al., 2016). According to the RNA1 and RNA2 sequences, the BBWV2 isolated in this study was most closely related to the BBWV2 isolate from Gynura procumbens (GenBank: KX686589) and the BBWV2 isolate from Nicotiana tabacum (GenBank: KX650868), respectively (Fig. S1E). To the best of our knowledge, this is the first report of I. balsamina naturally infected with BBWV2 in China. The study findings may be useful for detecting BBWV2 in I. balsamina and for diagnosing and managing the associated disease. The authors declare no conflict of interest. Yanhong Qiu and Haijun Zhang contributed equally to this paper. Funding: This research was supported by the Beijing Academy of Agriculture and Forestry Foundation, China (KYCX202305, QNJJ202131, and KJCX20230214). References: Qian H.Q., et al. 2023. J Ethnopharmacol. 303. Zheng Y., et al. 2022. Arch Virol. 167: 2099-2102. Kumar et al. 2016. Mol Biol Evol. 33: 1870-1874.

CmFSI8/CmOFP13 encoding an OVATE family protein controls fruit shape in melon.

Fruit shape is an important quality and yield trait in melon (Cucumis melo). Although some quantitative trait loci for fruit shape have been reported in in this species, the genes responsible and the underlying mechanisms remain poorly understood. Here, we identified and characterized a gene controlling fruit shape from two melon inbred lines, B8 with long-horn fruit and HP22 with flat-round fruit. Genetic analysis suggested that the shape was controlled by a single and incompletely dominant locus, which we designate as CmFSI8/CmOFP13. This gene was finely mapped to a 53.7-kb interval on chromosome 8 based on bulked-segregant analysis sequencing and map-based cloning strategies. CmFSI8/CmOFP13 encodes an OVATE family protein (OFP) and is orthologous to AtOFP1 and SlOFP20. The transcription level of CmFSI8/CmOFP13 in the ovary of HP22 was significantly higher than that in B8, and sequence analysis showed that a 12.5-kb genomic variation with a retrotransposon insertion identified in the promoter was responsible for elevating the expression, and this ultimately caused the differences in fruit shape. Ectopic overexpression of CmFSI8/CmOFP13 in Arabidopsis led to multiple phenotypic changes, including kidney-shaped leaves and shortened siliques. Taken together, our results demonstrate the involvement of an OFP in regulating fruit shape in melon, and our improved understanding of the molecular mechanisms will enable us to better manipulate fruit shape in breeding.

Melon shoot organization 1, encoding an AGRONAUTE7 protein, plays a crucial role in plant development.

KEY MESSAGE: A melon gene MSO1 located on chromosome 10 by map-based cloning strategy, which encodes an ARGONAUTE 7 protein, is responsible for the development of shoot organization. Plant endogenous small RNAs (sRNAs) are involved in various plant developmental processes. In Arabidopsis, sRNAs combined with ARGONAUTE (AGO) proteins are incorporated into the RNA-induced silencing complex (RISC), which functions in RNA silencing or biogenesis of trans-acting siRNAs (ta-siRNAs). However, their roles in melon (Cucumis melo L.) are still unclear. Here, the melon shoot organization 1 (mso1) mutant was identified and shown to exhibit pleiotropic phenotypes in leaf morphology and plant architecture. Positional cloning of MSO1 revealed that it encodes a homologue of Arabidopsis AGO7/ZIPPY, which is required for the production of ta-siRNAs. The AG-to-C mutation in the second exon of MSO1 caused a frameshift mutation and significantly reduced its expression. Ectopic expression of MSO1 rescued the Arabidopsis ago7 phenotype. RNA-seq analysis showed that several genes involved in transcriptional regulation and plant hormones were significantly altered in mso1 compared to WT. A total of 304 and 231 miRNAs were identified in mso1 and WT by sRNA sequencing, respectively, and among them, 42 known and ten novel miRNAs were differentially expressed. cme-miR390a significantly accumulated, and the expression levels of the two ta-siRNAs were almost completely abolished in mso1. Correspondingly, their targets, the ARF3 and ARF4 genes, showed dramatically upregulated expression, indicating that the miR390-TAS3-ARF pathway has conserved roles in melon. These findings will help us better understand the molecular mechanisms of MSO1 in plant development in melon.

First report of tomato spotted wilt virus infecting water dropwort in China.

Water dropwort (Oenanthe javanica) is an aquatic perennial plant that has been cultivated in many regions in Asia for thousands of years. In China, it is an economically important vegetable that has been consumed as food, while also being used as a folk remedy to alleviate diseases (Liu et al., 2021). In 2021, during a disease survey of a greenhouse in Beijing, China, chlorotic spots were detected on many water dropwort plants (Fig. S1A). Twenty-seven water dropwort samples were collected for the extraction of total RNA using the TRIzol reagent (Invitrogen, USA). High-quality RNA samples from three water dropwort plants were combined and used as the template for constructing a single small RNA library (BGI-Shenzhen Company, China). The Velvet 1.0.5 software was used to assemble the clean reads (18 to 28 nt) into larger contigs, which were then compared with the nucleotide sequences in the National Center database using the BLASTn algorithm. Thirty-eight contigs matched sequences in the tomato spotted wilt virus (TSWV) genome. No other viruses were detected. Twenty-seven leaf samples were analyzed in an enzyme-linked immunosorbent assay (ELISA) with anti-TSWV antibody (Agdia, USA), which revealed 17 positive reaction. Two sets of primer pairs targeting different parts of the S RNA (Table S1) was used to verify the TSWV infection on water dropwort by reverse transcription (RT)-PCR followed by Sanger sequencing (BGI-Shenzhen, China). The TSWV target sequences were amplified from 17 samples, which was consistent with the ELISA results. The sequenced 861-bp PCR product shared 99.8% nucleotide sequence identity with TSWV isolate MR-01 (MG593199), while the 441-bp amplicon shared a 99.2% nucleotide sequence identity with MR-01 (MG593199). To obtain the whole genome sequence of TSWV (S, M, and L RNA sequences), specific RT-PCR primers were designed (Table S1) and used to amplify their respective fragments from one representative sample (TSWV-water dropwort). The amplified products were inserted into PCE2TA/Blunt-Zero vector (Vazyme Biotech Co., Ltd, China) and then sequenced (BGI-Shenzhen, China). The S, M, and L RNA sequences were determined to be 2,952 nt (accession no. OM154969), 4,776 nt (accession no. OM154970), and 8,914 nt (accession no. OM154971), respectively. BLASTn analysis demonstrated that the whole genome sequence was highly conserved. The nucleotide identities between this isolate and other TSWV isolates ranged from 98.6% to 99.6% (S RNA), 98.9% to 99.2% (M RNA), and 97.3% to 98.7% (L RNA). Using MEGA 7.0, the phylogenetic relationships of TSWV were determined on the basis of the S, M, and L RNA full-length sequences (Kumar et al., 2016). In the S RNA derived phylogenetic tree, the water dropwort isolate was closely related to the MR-01 isolate from the USA (MG593199). In the M RNA and L RNA derived phylogenetic trees, the water dropwort isolate formed a branch with only a TSWV isolate from eggplant. Additionally, the M and L RNA sequences were most similar to sequences in TSWV isolates from China and Korea, respectively (Fig. S1B). To the best of our knowledge, this is the first report of water dropwort as a natural host for TSWV in China and the second report worldwide since the first finding in the Korea (Kil et al. 2020). TSWV has caused serious problems on many crops in the world, and the infection of TSWV on water dropwort in a greenhouse should not be looked lightly. Firstly, the virus can be passed on from generation to generation in infected water dropwort due to the vegetative propagation mode of the plant in production, thus threaten the production of this vegetable crop. In addition, infected water dropwort may serve as a reservoir for the virus, thus potentially posing a threat for causing TSWV spread in the affected greenhouses. The author(s) declare no conflict of interest. Funding: This research was supported by the Beijing Academy of Agriculture and Forestry Foundation, China (QNJJ202131, KJCX20200212, and KJCX20200113). References: Kil et al. 2020. Plant Pathol. J. 36: 67-75 Kumar et al. 2016. Mol Biol Evol, 33: 1870-1874 Liu et al. 2021. Horticulture Research. 8:1-17.

First report of squash leaf curl China virus infecting tomato in China.

Squash leaf curl China virus (SLCCNV) is a species in the genus Begomovirus that possess a bipartite genome. It is transmitted by the whitefly species Bemisia tabaci and infects cucurbit crops in various parts of the Old World (Wu et al., 2020). In 2020, tomato plants with curled, distorted and yellow leaves were found in a greenhouse in Shouguang, Shandong Province, China (Fig. S1). Leaves with these symptoms were collected from 11 plants and the total RNA was extracted with TRIzol reagent (Invitrogen, USA). Five RNA extracts of the highest quality were combined and a small RNA library was generated by the company (BGI-Shenzhen, China). About 22,338,920 clean reads (18-28nt) were acquired and assembled into larger contigs with the software Velvet 1.0.5. These were further compared against nucleotide sequences in the National Center for Biotechnology Information (NCBI) databases with BLASTn searches. Not unexpectedly, there were many assembled contigs that had high identities (90%-100% identities) with known tomato-infecting viruses, including 241 contigs matching tomato chlorosis virus, 26 contigs matching southern tomato virus, and 4 contigs matching tomato yellow leaf curl virus. However, 12 contigs had high identities (90%-100%) with the genomic DNA-A of SLCCNV, while 9 other contigs had high identities (90%-100%) with the genomic DNA-B of SLCCNV. To verify the presence of SLCCNV in tomato plants, two sets of primer pairs were designed according to the specific contigs assembled from derived small interfering RNAs (vsiRNAs). The primer pairs A742-F/A742-R (5'-GTAATACGAGCATCCGCACGGTAG-3'/5'-CGTGGAGGGCGAC AAACAGCTAACG-3') and B539-F/B539-R (5'-GCTACTTTCAAGGACGAAGAAGAGG-3'/5'-CG ACATAGATTTCTGGTCGGTGGGC-3') directed the amplification of 742 bp and 539 bp for DNA-A and DNA-B fragments, respectively, from the total genomic DNA of the 11 tomato samples. The DNA-A and DNA-B of SLCCNV were both detected from all of the tomato samples. After sequencing, the 742 bp PCR products shared 100% nucleotide sequence identity with the DNA-A of SLCCNV isolate GDXW (MW389919), whereas the PCR-amplified 539 bp fragments shared 100% nucleotide sequence identity with the DNA-B of SLCCNV isolate GDXW (MW389920). The full-length of DNA-A and DNA-B components were amplified with back-to-back primers A-F/A-R (Wu et al., 2020) and B-F/B-R (5'-GATAAACACGTCTCATTGCACCGC-3'/5'-GAGACGTGTTTATCAATATGGA CG-3'), respectively. The amplified fragments were further cloned into the PCE2TA/Blunt-Zero vector (Vazyme Biotech Co., China). After sequencing, the complete sequence of DNA-A was 2736 nt in length (MZ682117), while the DNA-B was 2718 nt in length (OK236348). The phylogenetic relationships of the DNA-A and DNA-B components were determined using MEGA 7 based on the full-length sequences of DNA-A and DNA-B, respectively (Kumar et al., 2016). Results showed that the DNA-A formed an independent cluster and was mostly related to the GDHY (MW389917) in the phylogenetic tree constructed using the neighbor-joining (NJ) method, while the DNA-B formed an independent cluster and was mostly related to the SLCCNV isolate BLDG (MW389928) and isolate GDBL (MW389922) (Fig. S2). The nt identities of DNA-A were also calculated with SDT v1.2 by comparison with other begomovirus sequences from the initial BLASTn analysis (Muhire et al., 2014), showing that the virus shared 99.4% sequence identity with SLCCNV isolate GDHY (MW389917). According to the current demarcation threshold for begomoviruses, recommended by the International Committee on Taxonomy of Viruses (ICTV) (91% nt identity) (Brown et al., 2015), this virus identified from tomato is a distinct strain of SLCCNV, designated SLCCNV-SDSG. To the best of our knowledge, this is the first report of a natural infection of SLCCNV on tomato in China. SLCCNV has caused serious problems in cucurbit production in some areas, so it will be important to investigate if tomato plays a role in the disease biology by serving as a reservoir host. The author(s) declare no conflict of interest. Funding: The funding for this research was supported by the Beijing Academy of Agriculture and Forestry Foundation, China (QNJJ202131, QNJJ201915, KJCX20200113). References: Brown et al. 2015. Arch Virol 160: 1593-1619 Kumar et al. 2016. Mol Biol Evol, 33: 1870-1874 Muhire et al. 2014. Plos One, 9 Wu et al. 2020. J Integr Agr, 19: 570-577.

Blue Native/SDS-PAGE and iTRAQ-Based Chloroplasts Proteomics Analysis of Nicotiana tabacum Leaves Infected with M Strain of Cucumber Mosaic Virus Reveals Several Proteins Involved in Chlorosis Symptoms.

Virus infection in plants involves necrosis, chlorosis, and mosaic. The M strain of cucumber mosaic virus (M-CMV) has six distinct symptoms: vein clearing, mosaic, chlorosis, partial green recovery, complete green recovery, and secondary mosaic. Chlorosis indicates the loss of chlorophyll which is highly abundant in plant leaves and plays essential roles in photosynthesis. Blue native/SDS-PAGE combined with mass spectrum was performed to detect the location of virus, and proteomic analysis of chloroplast isolated from virus-infected plants was performed to quantify the changes of individual proteins in order to gain a global view of the total chloroplast protein dynamics during the virus infection. Among the 438 proteins quantified, 33 showed a more than twofold change in abundance, of which 22 are involved in the light-dependent reactions and five in the Calvin cycle. The dynamic change of these proteins indicates that light-dependent reactions are down-accumulated, and the Calvin cycle was up-accumulated during virus infection. In addition to the proteins involved in photosynthesis, tubulin was up-accumulated in virus-infected plant, which might contribute to the autophagic process during plant infection. In conclusion, this extensive proteomic investigation on intact chloroplasts of virus-infected tobacco leaves provided some important novel information on chlorosis mechanisms induced by virus infection.

The cucumber mosaic virus movement protein suppresses PAMP-triggered immune responses in Arabidopsis and tobacco.

Cucumber Mosaic Virus (CMV) has a small RNA genome that encodes a limited number of proteins, but can infect many plant species, including Arabidopsis thaliana and Nicotiana benthamiana. Virus proteins thus have multiple means of conferring their pathogenicity during the infection process. However, the pathogenic mechanism of CMV remains unclear. Here we discovered that the expression of the CMV movement protein (MP) in A. thaliana and N. benthamiana can suppress reactive oxygen species (ROS) production triggered by multiple pathogen-associated molecular patterns (PAMPs), such as bacteria-derived peptide flg22, elf18, and fungal-derived chitin. Transgenic Arabidopsis plants expressing the MP were compromised in flg22-induced immune activation and were more susceptible to Pseudomonas syringae pv. tomato (Pst) DC3000 hrcC- strain infection. Further analysis revealed that flg22-induced resistance gene expression was also compromised in MP transgenic plants. The CMV MP protein was previously reported to function in cell-to-cell movement processes, and our findings offer a new molecular mechanism for the CMV MP protein in suppression of host PAMP-triggered immune responses.

Characterization of siRNAs derived from cucumber mosaic virus in infected tobacco plants.

This study characterized the viral small interfering RNAs (vsiRNAs) from Nicotiana tabacum cv. Samsun infected with a cucumber mosaic virus (CMV) 2b-deficient mutant. Most vsiRNAs were 21 -22 nucleotides in length and the 5'-terminal ends were dominated by A and U, respectively. The observed vsiRNAs were heterogeneously distributed throughout the CMV genome; however, most of the vsiRNAs were derived from sense strands, as opposed to antisense strands. These results demonstrate the conserved and specific function of Dicer-like (DCL), Argonaute (AGO) and RNA-dependent RNA polymerase (RDR) proteins in tobacco. Finally, it was revealed that vsiRNAs target abundant host genes, indicating complex roles for CMV vsiRNAs during the development of symptoms.

Virus infection triggers widespread silencing of host genes by a distinct class of endogenous siRNAs in Arabidopsis.

Antiviral immunity controlled by RNA interference (RNAi) in plants and animals is thought to specifically target only viral RNAs by the virus-derived small interfering RNAs (siRNAs). Here we show that activation of antiviral RNAi in Arabidopsis plants is accompanied by the production of an abundant class of endogenous siRNAs mapped to the exon regions of more than 1,000 host genes and rRNA. These virus-activated siRNAs (vasiRNAs) are predominantly 21 nucleotides long with an approximately equal ratio of sense and antisense strands. Genetically, vasiRNAs are distinct from the known plant endogenous siRNAs characterized to date and instead resemble viral siRNAs by requiring Dicer-like 4 and RNA-dependent RNA polymerase 1 (RDR1) for biogenesis. However, loss of exoribonuclease4/thylene-insensitive5 enhances vasiRNA biogenesis and virus resistance without altering the biogenesis of viral siRNAs. We show that vasiRNAs are active in directing widespread silencing of the target host genes and that Argonaute-2 binds to and is essential for the silencing activity of vasiRNAs. Production of vasiRNAs is readily detectable in Arabidopsis after infection by viruses from two distinct supergroups of plant RNA virus families and is targeted for inhibition by the silencing suppressor protein 2b of Cucumber mosaic virus. These findings reveal RDR1 production of Arabidopsis endogenous siRNAs and identify production of vasiRNAs to direct widespread silencing of host genes as a conserved response of plants to infection by diverse viruses. A possible function for vasiRNAs to confer broad-spectrum antiviral activity distinct to the virus-specific antiviral RNAi by viral siRNAs is discussed.

Activation of a plasmid-situated type III PKS gene cluster by deletion of a wbl gene in deepsea-derived Streptomyces somaliensis SCSIO ZH66.

BACKGROUND: Actinomycete genome sequencing has disclosed a large number of cryptic secondary metabolite biosynthetic gene clusters. However, their unavailable or limited expression severely hampered the discovery of bioactive compounds. The whiB-like (wbl) regulatory genes play important roles in morphological differentiation as well as secondary metabolism; and hence the wblA so gene was probed and set as the target to activate cryptic gene clusters in deepsea-derived Streptomyces somaliensis SCSIO ZH66. RESULTS: wblA so from deepsea-derived S. somaliensis SCSIO ZH66 was inactivated, leading to significant changes of secondary metabolites production in the ΔwblA so mutant, from which α-pyrone compound violapyrone B (VLP B) was isolated. Subsequently, the VLP biosynthetic gene cluster was identified and characterized, which consists of a type III polyketide synthase (PKS) gene vioA and a regulatory gene vioB; delightedly, inactivation of vioB led to isolation of another four VLPs analogues, among which one was new and two exhibited improved anti-MRSA (methicillin-resistant Staphylococcus aureus, MRSA) activity than VLP B. Moreover, transcriptional analysis revealed that the expression levels of whi genes (whiD, whiG, whiH and whiI) and wbl genes (wblC, wblE, wblH, wblI and wblK) were repressed by different degrees, suggesting an intertwined regulation mechanism of wblA so in morphological differentiation and secondary metabolism of S. somaliensis SCSIO ZH66. CONCLUSIONS: wblA orthologues would be effective targets for activation of cryptic gene clusters in marine-derived Streptomyces strains, notwithstanding the regulation mechanisms might be varied in different strains. Moreover, the availability of the vio gene cluster has enriched the diversity of type III PKSs, providing new opportunities to expand the chemical space of polyketides through biosynthetic engineering.

A New Dioic Acid from a wbl Gene Mutant of Deepsea-Derived Streptomyces somaliensis SCSIO ZH66.

The wblAso gene functions as a global regulatory gene in a negative manner in deepsea-derived Streptomyces somaliensis SCSIO ZH66. A new dioic acid (1) as well as two known butenolides (2 and 3) were isolated from the ΔwblAso mutant strain of S. somaliensis SCSIO ZH66. The structure of 1 was elucidated by a combination of spectroscopic analyses, including MS and NMR techniques. In the cell growth inhibitory evaluation, compound 3 exhibited moderate activity against the human hepatic carcinoma cell line (Huh7.5) with an IC50 value of 19.4 µg/mL, while compounds 1 and 2 showed null activity up to 100 µg/mL.

The detection of hydrogen peroxide involved in plant virus infection by fluorescence spectroscopy.

The production of reactive oxygen species (ROS) forms part of the defense reaction of plants against invading pathogens. ROS have multifaceted signaling functions in mediating the establishment of multiple responses. To verify whether hydrogen peroxide (H2 O2 ) contributes to plant virus infection and the development of induced symptoms, we used fluorescence to monitor the generation of H2 O2 and confocal laser scanning microscopy (CLSM) to investigate the subcellular distribution of H2 O2 in leaves. In this study, the M strain of Cucumber mosaic virus (M-CMV) induced heavy chlorotic symptoms in Nicotiana tabacum cv. white burley during systemic infection. Compared with mock-inoculated leaves, H2 O2 accumulation in inoculated leaves increased after inoculation, then decreased after 4 days. For systemically infected leaves that showed chlorotic symptoms, H2 O2 accumulation was always higher than in healthy leaves. Subcellular H2 O2 localization observed using CLSM showed that H2 O2 in inoculated leaves was generated mainly in the chloroplasts and cell wall, whereas in systemically infected leaves H2 O2 was generated mainly in the cytosol. The levels of coat protein in inoculated and systemically infected leaves might be associated with changes in the level of H2 O2 and symptom development. Further research is needed to elucidate the generation mechanism and the relationship between coat protein and oxidative stress during infection and symptom development. Copyright © 2016 John Wiley & Sons, Ltd.

Rapid detection of the pathogenic fungi causing blackleg of Brassica napus using a portable real-time fluorescence detector.

The fungus Leptosphaeria maculans leading to Phoma stem canker (blackleg) of Brassica napus (oilseed rape, canola) produces the phytotoxin sirodesmin PL, which is responsible for major yield losses of oilseed rape worldwide. Polymerase chain reaction (PCR) remains the gold standard diagnostic tool for L. maculans, but the required expensive equipment and long time make it inappropriate for fast field test. Herein, a portable system for rapid assaying L. maculans and L. biglobosa is designed around recombinase polymerase amplification (RPA) with fluorescent probe as the signal indicator, which allowed the real-time assay of amplification performed on a portable device between 37 and 42 °C. The time needed to observe the positive reaction results is controlled within 30 min. The proposed assay system is a good choice for on-site disease screening of oilseed rape plant where rapid detection is valuable, including port quarantine, agriculture quality testing, and pathogen spreading control.

Profiling of small RNAs derived from cucumber mosaic virus in infected Nicotiana benthamiana plants by deep sequencing.

In plants, RNA silencing is a conserved mechanism underlying antiviral immunity. To investigate antiviral responses in Nicotiana benthamiana, we analyzed the profiles of the virus-derived small RNAs (vsRNAs) in wild-type N. benthamiana and NbRDR6 mutant plants infected with the cucumber mosaic virus (CMV) 2b-deficient mutant. We observed that NbRDR6 regulates RNA silencing by producing vsRNAs that trigger an effective antiviral response, while NbRDR1 may nonredundantly and synergistically function with NbRDR6 to mediate immune responses. The vsRNAs in N. benthamiana and NbRDR6 mutant plants mainly comprised 21 or 22 nucleotides, and mostly consisted of a 5'-terminal adenine. Additionally, NbAGO2 expression was significantly up-regulated in N. benthamiana and NbRDR6 mutant plants, suggesting that NbAGO2 is closely associated with the antiviral activities of vsRNAs. The distribution of vsRNAs in the CMV genome was biased toward RNA sense strands in both N. benthamiana and NbRDR6 mutant plants. These findings indicate the specific and conserved antiviral immunity in Nicotiana benthamiana.

Cucumber mosaic virus coat protein induces the development of chlorotic symptoms through interacting with the chloroplast ferredoxin I protein.

Cucumber mosaic virus (CMV) infection could induce mosaic symptoms on a wide-range of host plants. However, there is still limited information regarding the molecular mechanism underlying the development of the symptoms. In this study, the coat protein (CP) was confirmed as the symptom determinant by exchanging the CP between a chlorosis inducing CMV-M strain and a green-mosaic inducing CMV-Q strain. A yeast two-hybrid analysis and bimolecular fluorescence complementation revealed that the chloroplast ferredoxin I (Fd I) protein interacted with the CP of CMV-M both in vitro and in vivo, but not with the CP of CMV-Q. The severity of chlorosis was directly related to the expression of Fd1, that was down-regulated in CMV-M but not in CMV-Q. Moreover, the silencing of Fd I induced chlorosis symptoms that were similar to those elicited by CMV-M. Subsequent analyses indicated that the CP of CMV-M interacted with the precursor of Fd I in the cytoplasm and disrupted the transport of Fd I into chloroplasts, leading to the suppression of Fd I functions during a viral infection. Collectively, our findings accentuate that the interaction between the CP of CMV and Fd I is the primary determinant for the induction of chlorosis in tobacco.

Epigenetic Changes in the Regulation of Nicotiana tabacum Response to Cucumber Mosaic Virus Infection and Symptom Recovery through Single-Base Resolution Methylomes.

Plants have evolved multiple mechanisms to respond to viral infection. These responses have been studied in detail at the level of host immune response and antiviral RNA silencing (RNAi). However, the possibility of epigenetic reprogramming has not been thoroughly investigated. Here, we identified the role of DNA methylation during viral infection and performed reduced representation bisulfite sequencing (RRBS) on tissues of Cucumber mosaic virus (CMV)-infected Nicotiana tabacum at various developmental stages. Differential methylated regions are enriched with CHH sequence contexts, 80% of which are located on the gene body to regulate gene expression in a temporal style. The methylated genes depressed by methyltransferase inhibition largely overlapped with methylated genes in response to viral invasion. Activation in the argonaute protein and depression in methyl donor synthase revealed the important role of dynamic methylation changes in modulating viral clearance and resistance signaling. Methylation-expression relationships were found to be required for the immune response and cellular components are necessary for the proper defense response to infection and symptom recovery.

Rapid Detection of Prunus Necrotic Ringspot Virus by Reverse Transcription-cross-priming Amplification Coupled with Nucleic Acid Test Strip Cassette.

Prunus necrotic ringspot virus (PNRSV) is one of the most devastating viruses to Prunus spp. In this study, we developed a diagnostic system RT-CPA-NATSC, wherein reverse transcription-cross-priming amplification (RT-CPA) is coupled with nucleic acid test strip cassette (NATSC), a vertical flow (VF) visualization, for PNRSV detection. The RT-CPA-NATSC assay targets the encoding gene of the PNRSV coat protein with a limit of detection of 72 copies per reaction and no cross-reaction with the known Prunus pathogenic viruses and viroids, demonstrating high sensitivity and specificity. The reaction is performed on 60 °C and can be completed less than 90 min with the prepared template RNA. Field sample test confirmed the reliability of RT-CPA-NATSC, indicating the potential application of this simple and rapid detection method in routine test of PNRSV.

Transcriptome analysis of Nicotiana tabacum infected by Cucumber mosaic virus during systemic symptom development.

Virus infection of plants may induce a variety of disease symptoms. However, little is known about the molecular mechanism of systemic symptom development in infected plants. Here we performed the first next-generation sequencing study to identify gene expression changes associated with disease development in tobacco plants (Nicotiana tabacum cv. Xanthi nc) induced by infection with the M strain of Cucumber mosaic virus (M-CMV). Analysis of the tobacco transcriptome by RNA-Seq identified 95,916 unigenes, 34,408 of which were new transcripts by database searches. Deep sequencing was subsequently used to compare the digital gene expression (DGE) profiles of the healthy plants with the infected plants at six sequential disease development stages, including vein clearing, mosaic, severe chlorosis, partial and complete recovery, and secondary mosaic. Thousands of differentially expressed genes were identified, and KEGG pathway analysis of these genes suggested that many biological processes, such as photosynthesis, pigment metabolism and plant-pathogen interaction, were involved in systemic symptom development. Our systematic analysis provides comprehensive transcriptomic information regarding systemic symptom development in virus-infected plants. This information will help further our understanding of the detailed mechanisms of plant responses to viral infection.