China is the center of origin of kiwifruit (Actinidia spp.) and the largest producing country in both terms of yield and acreage (Ferguson 2015). Kiwifruit virus diseases usually cause leaf yellowing, mosaic spots, fruit malformation, resulting in reduced yield and quality (Blouin et al. 2013). In August 2019, virus-like symptoms including chlorotic spots, crinkle, yellowing, and deformation were observed on leaves of kiwifruit grown in a 2,000-m2 commercial orchard in Chongqing, China, with disease incidence reaching approximately 20%. Twenty-eight leaf samples (CQ1-28) were randomly collected from symptomatic plants and mixed in equal amounts. Extraction of total RNA from above pooled sample, removal of ribosomal RNA and subsequent high-throughput sequencing (HTS) were conducted as previously reported (Wen et al. 2020). After filtering low-quality reads (Q30>94.16%), a total of 82,730,664 clean reads with an average length of 150 nucleotides (nt) were obtained and de novo assembled into contigs using CLC Genomics Workbench 11.0 (Qiagen) with k-mer values ranging from 31 to 121. BLASTx against the NCBI GenBank database showed that 10 contigs, 344 to 2,905 nt in length, were annotated as apple latent spherical virus (ALSV) (AB030940.1 and AB030941.1), which belongs to the Cheravirus genus and was originally identified in apple (Li et al. 2000). The nt identity between these contigs and the reference genome ranged from 75.2 to 85.7%. Primers CP-F/CP-R (5′-RTAGGHCARGGKGCTTGYTTGAGTA-3′/5′-CCAATAGGCTGTAGTRCTGGCC ATA-3′) targeting a 750 bp fragment CP gene of ALSV were designed based on the obtained contigs and the AB030941.1 sequence to verify the sequencing results in the individual samples. Total RNA was individually extracted from leaf samples CQ1-28 using a cetyltrimethylammonium bromide (CTAB) based method (Li et al., 2008) and subjected to reverse transcription polymerase chain reaction (RT-PCR). Target products were obtained from four samples (CQ2, CQ6, CQ8 and CQ15) and Sanger sequenced. Alignment of the four sequences showed that they shared 90.4 to 100% nt identity with each other and 75.7 to 77.3% nt identity with the corresponding AB030941.1 sequence. Furthermore, 49 symptomatic leaf samples were collected from Hubei (6), Jiangxi (6), Henan (31), Shanxi (4) provinces and Shanghai (2), China, and were tested for the presence of ALSV by RT-PCR with primers CP-F/CP-R. Three additional samples (Z2, ZZ19 and ZZ21) from Henan province were found ALSV positive. The detection rate of ALSV in Chongqing city and Henan province was 14.3% and 9.7%, respectively. To analyze the phylogenetic relationship of the ALSV isolates identified in this study, the seven CP sequences (GenBank accession nos. PV931796.1 to PV931802.1) of ALSV-positive kiwifruit samples together with the available sequences of ALSV from apple and Angelica sinensis (OP038546.1) (Jin et al. 2023) were aligned and a phylogenetic tree was constructed. The ALSV isolates obtained from kiwifruit clustered in the same branch with those infecting apple and A. sinensis. Since the mixed infections of ALSV with one or more of Actinidia virus 1, citrus leaf blotch virus, Actinidia virus A, Actinidia virus B and Actinidia seed borne latent virus, ALSV pathogenicity in kiwifruit remains challenging to confirm. To our knowledge, this is the first report of ALSV naturally infecting kiwifruit, expanding the host range of this virus and providing a crucial basis for viral disease monitoring.

Bacterial wilt (BW), caused by Ralstonia solanacearum, is a highly destructive disease in tomato, and resistance to BW is attenuated under high temperature (HT). However, limited information is available with respect to the molecular basis of tomato-R. solanacearum interactions under HT. Here, we conducted transcriptomic analysis on the root-stem junction tissues of tomatoes exhibiting varying levels of BW resistance at 0, 12, 24, and 48 h post-inoculation (hpi) with R. solanacearum under 33 °C. Weighted gene co-expression network analysis (WGCNA) revealed two modules containing key genes that participated in disease resistance under HT. A total of 91 core genes were identified as potentially coordinating immune signaling and metabolic homeostasis during the interactions between tomato and R. solanacearum. Ultimately, we identified HsfA9 as a key transcription factor potentially involved in bacterial wilt resistance under HT. Our findings provide a valuable resource for elucidating the molecular mechanism underlying of R. solanacearum-tomato interactions under HT, thereby facilitating the development of effective strategies in disease control and prevention.

Tomato (Solanum lycopersicum L.) is a globally important vegetable crop, and postharvest diseases cause serious losses during storage and transportation. In recent years, fruit decay was observed on stored tomato fruits in Guangzhou, Guangdong Province, China. Three fungal isolates (SL-NC1, SL-NC2, SL-NC3) were obtained and identified as Neopestalotiopsis cubana based on morphological characters and ITS-TUB2-TEF1 multilocus phylogeny analysis. Pathogenicity tests confirmed that the three isolates could quickly infect tomatoes and induce fruit rot, fulfilling Koch's postulates. To our knowledge, this is the first report of N. cubana causing postharvest fruit rot in tomatoes.

RNA silencing, a conserved gene-regulatory mechanism mediated by small interfering RNAs (siRNAs), is a major component of plant antiviral immunity. The plant RNA-binding protein SUPPRESSOR OF GENE SILENCING 3 (SGS3) forms condensates that drive siRNA body assembly and promote siRNA biogenesis however, the regulators of SGS3 condensate formation and their roles in antiviral responses remain largely unknown. Here, we show that the cold-regulated 15-kDa dehydrin protein (COR15) homologs from Citrus aurantifolia and Nicotiana benthamiana interact with the citrus tristeza virus (CTV)-encoded p20 protein. COR15 abolishes the RNA silencing suppressor activity of p20, enhances antiviral RNA silencing, and confers resistance to CTV in N. benthamiana. COR15 also restricts infection by other positive-sense RNA viruses, suggesting that it functions as a broad-spectrum antiviral factor. Mechanistically, CTV infection induces NbCOR15 expression and promotes the formation of COR15 granules in the cytoplasm. COR15 attenuates p20-mediated SGS3 degradation by disrupting the p20-SGS3 interaction, thereby stabilizing SGS3. COR15 is recruited into siRNA bodies and undergoes liquid-liquid phase separation within these structures. Notably, COR15 promotes SGS3 condensate formation and efficient siRNA processing. CRISPR-Cas9-mediated knockout of COR15 in N. benthamiana plants reduces the number of SGS3 condensates, leads to abnormally sized SGS3 condensates, and impairs SGS3-dependent siRNA synthesis, indicating that COR15 is a previously unrecognized component of siRNA bodies. Together, our findings reveal the function and mechanism of a dehydrin protein in plant antiviral immunity and provide new insights into the molecular arms race underlying plant-virus co-evolution.

Soil alkalinity severely limits the productivity of strawberry, a high-value horticultural crop. The root endophytic fungus Piriformospora indica enhances plant stress resilience, yet the systemic mechanisms underlying its promotion of alkaline tolerance remain poorly understood. This study employed an integrated transcriptomic and metabolomic approach to elucidate these mechanisms in strawberry under alkaline stress. Inoculation with P. indica significantly alleviated stress-induced growth inhibition, improving biomass accumulation and leaf development. Metabolomic profiling identified 1,352 DAMs, predominantly flavonoids and phenolic acids. Transcriptome analysis revealed 19,689 DEGs enriched in oxidoreductase activity, hormone signaling, and secondary metabolism. Multi-omics integration highlighted coordinated changes in the metabolism of pyruvate,alanine, aspartate and glutamate. P. indica maintained the balance of carbon and nitrogen allocation in strawberry under alkaline stress, an effect linked to the downregulation of argG, asnB, and GLT1. These findings suggest a putative systemic metabolic mechanism by which P. indica may enhance alkaline tolerance, potentially through rebalancing primary metabolism. This offers molecular insights into fungal-mediated stress adaptation in strawberry and supports its potential as a sustainable bio-inoculant for improving productivity in alkaline soils.

High-temperature treatment (thermotherapy) inhibits viral RNA synthesis, restricting viral particle movement into the apical meristem and enabling virus elimination when applied for optimized durations. Combined thermotherapy and meristem culture enhances the production of virus-free germplasm. Here, we describe optimized protocols for thermotherapy, meristem culture, and transplantation of virus-free pear plants.

Pear ring rot disease (Botryosphaeria dothidea) is a significant threat to the healthy development of the pear industry. Recent research has identified the functional role of long non-coding RNAs (lncRNAs) in various biological processes of plants. The role of lncRNAs in the pear defense response remains unknown. In this study, transcriptome sequencing was used to analyze lncRNAs in pear stem infected with B. dothidea. It identified 3555 lncRNAs, of which 286 were significantly differentially expressed. GO and KEGG analyses showed that cis- and trans-regulated target genes were enriched in multiple disease resistance-related pathways. More specifically, MSTRG.32189, predicted as an endogenous target mimic (eTM), was significantly down-regulated in response to B. dothidea infection, and was confirmed to inhibit the cleavage effect of PcmiR399b on PcUBC24. OE-MSTRG.32189 transgenic Arabidopsis exhibited lower Pi content and weaker disease resistance to Botrytis cinerea compared with wild type. In pear callus, overexpression of MSTRG.32189 negatively regulated PcmiR399b, which decreased Pi content and reduced disease resistance. Overexpressing PcmiR399b in pear callus exhibited the opposite effects compared with OE-MSTRG.32189. Overexpression and knockout of PcUBC24 further clarified that PcUBC24 negatively regulates Pi content and disease resistance to B. dothidea infection. Furthermore, the ROS levels and expressions of disease resistance pathway-related genes were regulated by the MSTRG.32189-PcmiR399b-PcUBC24 module in transgenic pear callus, which contributed to disease resistance. Overall, our results demonstrated the role of lncRNAs in the pear defense response, revealing that the MSTRG.32189-PcmiR399b-PcUBC24 module regulates phosphate accumulation and disease resistance to B. dothidea infection in pear.

Pear ring rot disease, the pathogen of Botryosphaeria dothidea causes significant threat to the healthy development of the pear industry, therefore the exploration of disease-resistant gene resources is crucial for disease prevention and control. Members of the R2R3-MYB subfamily play important roles in regulating pathogen resistance in plants, however the gene function in regulating host resistance in pear remains unclear. In this study, the role of PcMYB44 were investigated in regulating host resistance disease in pear calli using both forward and reverse genetic approaches. Overexpression of PcMYB44 positively regulates the disease resistance, whereas knockout of PcMYB44 results in a phenotype with decreased resistance. Our results further demonstrated that PcMYB44 could directly affect lignin content and resistance to fungal diseases by regulating the PcmiR397-PcLACs module and lignin biosynthesis gene expression levels. Additionally, overexpressing PcMYB44 also elevated expression levels of key genes of JA/SA/ET pathway. The obtained results revealed that PcMYB44 regulated host resistance to ring rot disease through synergistic regulation the lignification and activating disease-resistance gene expression of JA/SA/ET defense pathways as a underlying secondary mechanism, which provide valuable genetic resources for molecular breeding for disease resistance.

Viruses exploit autophagy to degrade host immune components for their successful infection. However, how viral factors sequester the autophagic substrates into autophagosomes remains largely unknown. In this study, we showed that p20 protein, a viral suppressor of RNA silencing (VSR) encoded by citrus tristeza virus (CTV), mediated autophagic degradation of SUPPRESSOR OF GENE SILENCING 3 (SGS3), a plant-specific RNA-binding protein that is pivotal in antiviral RNA silencing. CTV infection activated autophagy, and the overexpression of p20 was sufficient to induce autophagy. Silencing of autophagy-related genes NbATG5 and NbATG7 attenuated CTV infection in Nicotiana benthamiana plants. In contrast, knockdown of the autophagy negative-regulated genes NbGAPCs led to virus accumulation, indicating the proviral role of autophagy in CTV infection. Further investigation found that p20 interacted with autophagy-related protein ATG8 through two ATG8-interacting motifs (AIMs) and sequestered SGS3 into autophagosomes by forming the ATG8-p20-SGS3 ternary complex. The mutations of the two AIMs in p20 (p20mAIM1 and p20mAIM5) abolished the interaction of p20 with ATG8, resulting in the deficiency of autophagy induction, SGS3 degradation, and VSR activity. Consistently, N. benthamiana plants infected with mutated CTVmAIM1 and CTVmAIM5 showed milder symptoms and decreased viral accumulation. Taken together, this study uncovers the molecular mechanism underlying how a VSR mediates the interplay between RNA silencing and autophagy to enhance the infection of a closterovirus.

China is a major producer of pears in the world and anthracnose is the most important disease, which may include fruit rot and early defoliation, and further brings enormous economic losses. In August of 2023, a sudden outbreak of anthracnose disease, ranging from 70% to 90% disease incidence, occurred on fruits of Pyrus pyrifolia (Burm.f.) Nakai in 200 acres of three pear orchards at Baiyi town, Guiyang city, Guizhou Province of China. Thirty diseased fruits were randomly collected three pear orchards, i.e., 10 fruits from each, and transported to the laboratory for analysis. The symptoms showed dark brown lesions that extended rapidly causing sunken fruit rot lesions harboring orange conidial clumps. To identify the pathogen, three from the 10 diseased fruits per orchard were used for isolation. Small tissue pieces of diseased fruits were disinfected and rinsed twice with 75% alcohol and sterile water, placed on a potato dextrose agar (PDA), and cultured at 25℃ in a dark (Fang, 1998). Ten isolates with same colony morphology were obtained. Two isolates, GZ-3 and GZ-5, were randomly selected for further analysis. The GZ-3 and GZ-5 isolates grew on PDA plates at an average daily growth rate of 0.8~1.0 cm/d in the darkness at 25℃. The colonies were white to gray on the front side and greyish-green to orange on the reverse side. The conidia were hyaline, smooth, nonseptate, and fusiform, measuring 9.0-19.2×2.5-6.1 μm, with the average mean of 13.9±2.2×4.4±0.9 μm (n=50) for GZ-3, and the size of 9.5-15.4×2.3-4.8 μm, with average size of 13.1±1.5×3.7±0.5 μm (n=50) for GZ-5, respectively. The morphological characteristics of the GZ-3 and GZ-5 colonies were consistent with those of Colletotrichum acutatum complex (Damm et al. 2012). To confirm the pathogenicity, the 6 × 106 mL-1 spore suspension of GZ-3 and GZ-5 were inoculated on healthy P. pyrifolia (Burm.f.) Nakai fruits using the non-invasive spray method, while sterile water was used to inoculate control fruits. The fruits inoculated with the two isolates exhibited dark brown spots in the early stage of the disease, then the lesions expanded to rot and produced orange conidia. GZ-3 and GZ-5 were reisolated from the fruits, fulfilling Koch's postulates. The internal transcribed spacer (ITS) rDNA, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin (ACT), chitin synthase 1 (CHS-1) and β-tubulin (TUB2) were partially amplified using the primers designed previously (Weir et al. 2012) and sequenced. The above five gene sequences from GZ-3 showed 99.63% to 100% identity with those of GZ-5, respectively. The GZ-3 sequences were deposited in GenBank (ACT: PP825836; TUB2: PP825837; CHS-1: PP825838; GAPDH: PP825839; ITS: PP821112). The sequence alignment was performed based on the concatenated sequence of ACT-TUB2-CHS-1-GAPDH-ITS. A phylogenetic tree, constructed using the Neighbor-Joining method with bootstrap replication 2200 bootstrap alignments in MEGA11.0, showed GZ-3 and GZ-5 clustering with reference C. nymphaeae isolates. Based on the obtained morphological characterization and phylogenetic analysis results, GZ-3 and GZ-5 were identified as C. nymphaeae. Currently, 12 Colletotrichum species causing pear anthracnose have been reported in China (Fu et al. 2019). To our knowledge, this is the first report of C. nymphaeae causing pear anthracnose in the country. The findings are significant for a quick monitoring and control of pear anthracnose.

ABSTRACT: Seed vigor of tobacco (Nicotiana tabacum L.) is established during seed development stage, while its regulators remain largely unknown. Here, a comparative peptidomics analysis of the developing seeds was conducted to reveal the regulators involving the establishment of tobacco seed vigor. The most significant difference of seed vigor was observed between seeds harvested at 20 and 30 days after pollination (DAP), and then the corresponding seeds were collected separately for peptidomics analysis. A total of 2932 and 2812 nonredundant peptides were identified in seeds harvested at 20 and 30 DAP, respectively. In which, 349 differentially expressed peptides (DEPs) were characterized. To explore the potential functions of these DEPs, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were further analyzed according to their precursor proteins. Most DEP precursor proteins were involved in response to abiotic stimulus, response to water, and protein processing in endoplasmic reticulum. Further, the peptides derived from the precursor proteins, such as late embryogenesis abundant (LEA) protein, heat shock protein, peroxiredoxin, and globulin proteins, may regulates the establishment of seed vigor by influencing reactive oxygen species. The results provide a foundation for further exploration of the peptides functions on the establishment of seed vigor in tobacco.

Pear is popular among people, which is an important pillar industry in China. In March of 2023, dark brown necrotic lesions were discovered on the trunks of Pyrus pyrofolia cv. Osmanthus pear in orchard, Liuzhou City, Guangxi Zhuang Autonomous Region. In August, field investigation and sample collection were conducted in orchard. Forty pear trees were selected for symptomatic observation, which of 21 had lesions ranging from 10 to 24 per tree, and 19 with 1 to 8 lesions, respectively. To isolate the pathogen, small tissue pieces of 3 diseased pear trunk samples were disinfected with 75% ethanol for 1 minute, rinsed with sterile water, and dried with filter paper. The tissue pieces were placed on potato dextrose agar (PDA) plates and cultured in a dark incubator at 25℃. Six isolates with the similar morphology were obtained. One of the six isolates was randomly selected as the representative strain and named as GX-3. Mycelium grows with an average rate of 4.26 cm/d. The hypha is highly aerial, and is initially white and then turns black. Subsequently, pycnidia formed and secreted black mucus on the PDA medium after 28 days. The immature conidia were ellipsoid, colorless, hyaline, and unicellular, mostly becoming brown bicellular with longitudinal stripes at maturity. The conidial size was 22.5 to 32.6×12.1 to 19.7μm, and the average size was 28.4±2.3×16.7±2.0 μm (n=50), respectively. GX-3 colony morphology was consistent with that of Lasiodiplodia pseudotheobromae (Alves et al.2008). For molecular identification, the internal transcribed spacer of rDNA (ITS), translation elongation factor 1-α (TEF1-α), and β-tubulin regions were amplified using the primers ITS1/4, EF1-728F/986R, and Bt2a/Bt2b, respectively (White et al.1990; Carbone and Kohn 1999; Glass and Donaldson 1995). The obtained sequences of GX-3 were deposited in NCBI with Accession numbers OR655421, OR661231, and OR661230, respectively. The sequences of ITS, TEF1-α, and β-tubulin from GX-3 are 99.44%、99.67% and 99.78% identities with those of L. pseudotheobromae CBS 447.62, respectively. The phylogenetic analysis was performed by maximum likelihood method, revealing that GX-3 is closely clustered with the isolates of L. pseudotheobromae. Therefore, the GX-3 strain was identified as L. pseudotheobromae. GX-3 was further analyzed for its pathogenicity on pear. Firstly, the GX-3 mycelium plugs and spraying spore suspension with the concentration of 1×107 conidia/ml were applied on the stems of 4-month-old healthy birch-leaf pear (Pyrus betulifolia Bunge) potted seedlings by acupuncture needle method, meanwhile PDA and sterile water were used as controls. After 3 days of inoculation, stem surface of the birch-leaf pear exhibited dark brown lesions with slight surface depression, obvious dryness, and canker symptoms, while the control treatment showed no symptoms. The GX-3 was also inoculated on in vitro branches of 'Hosui', 'Hongxiangsu', 'Bodoqing' and 'Xuehua', showing dark brown canker lesions. The same pathogen can be successfully isolated from diseased stems and branches but not from the controls, which accomplishes Koch's postulates. L. pseudotheobromae has been widely reported that it can cause rot and canker on apple, walnut, hackberry, and so on (Xue et al. 2019; Wang et al. 2023; Liang et al. 2020). This is the first report of necrosis and canker disease caused by L. pseudotheobromae on pear in China, which is a potential threat to pear industry.

Pear black spot (Alternaria spp.) disease widespread in most pear production regions in China, causes early defoliation, which leads to the decline of fruit quality and heavily restricts the healthy development of the pear industry. Presently, no efficient way was found to biocontrol pear black spot disease. The mycovirus-mediated hypovirulent strain is regarded as an important measure of biocontrol of the fungal disease. Therefore, it is particularly urgent to excavate mycoviral resources with hypovirulence in Alternaria spp. strains. This study obtained partial or complete genomes of 14 mycoviruses from 83 Alternaria spp. strains from Wuhan city of China by high throughput sequencing, including 3 double-stranded (ds) RNA and 11 positive single-stranded (+ss) types, which were grouped into 8 distinct lineages, belonging to Chrysoviridae (isolate of AaCV1), Curvulaviridae (one virus), Partitiviridae (one virus), Mitoviridae (three viruses), Botourmiaviridae (five viruses), Deltaflexiviridae (one virus), Fusariviridae (one virus) and Togaviridae (one virus). The multiple mycoviral coinfections were common and prevalent, accounting for 78 % (65/83), with each strain harboring different numbers of mycoviruses, ranging from two to seven in Alternaria spp. Furthermore, the effects of the mycoviruses on the host were analyzed to screen the viruses-inducing hypovirulent strains. The derivative isolates from hypovirulent HB-145 with different mycoviruses were obtained by vertical transmission. It revealed that the mycoviruses have different effects on the virulence of derivative isolates from HB-145. In conclusion, these findings provide important molecular information for the classification, origin, and genetic variety of mycoviruses, and candidate materials for the exploitation of mycoviruses as biocontrol agents.

Circular single-stranded DNA (ssDNA) viruses have been rarely found in fungi, and the evolutionary and ecological relationships among ssDNA viruses infecting fungi and other organisms remain unclear. In this study, a novel circular ssDNA virus, tentatively named Diaporthe sojae circular DNA virus 1 (DsCDV1), was identified in the phytopathogenic fungus Diaporthe sojae isolated from pear trees. DsCDV1 has a monopartite genome (3185 nt in size) encapsidated in isometric virions (21-26 nm in diameter). The genome comprises seven putative open reading frames encoding a discrete replicase (Rep) split by an intergenic region, a putative capsid protein (CP), several proteins of unknown function (P1-P4), and a long intergenic region. Notably, the two split parts of DsCDV1 Rep share high identities with the Reps of Geminiviridae and Genomoviridae, respectively, indicating an evolutionary linkage with both families. Phylogenetic analysis based on Rep or CP sequences placed DsCDV1 in a unique cluster, supporting the establishment of a new family, tentatively named Gegemycoviridae, intermediate to both families. DsCDV1 significantly attenuates fungal growth and nearly erases fungal virulence when transfected into the host fungus. Remarkably, DsCDV1 can systematically infect tobacco and pear seedlings, providing broad-spectrum resistance to fungal diseases. Subcellular localization analysis revealed that DsCDV1 P3 is systematically localized in the plasmodesmata, while its expression in trans-complementation experiments could restore systematic infection of a movement-deficient plant virus, suggesting that P3 is a movement protein. DsCDV1 exhibits unique molecular and biological traits not observed in other ssDNA viruses, serving as a link between fungal and plant ssDNA viruses and presenting an evolutionary connection between ssDNA viruses and fungi. These findings contribute to expanding our understanding of ssDNA virus diversity and evolution, offering potential biocontrol applications for managing crucial plant diseases.

Pear chlorotic leaf spot-associated virus (PCLSaV) is a newly described emaravirus that infects pear trees. The virus genome consists of at least five single-stranded, negative-sense RNAs. The P5 encoded by RNA5 is unique to PCLSaV. In this study, the RNA silencing suppression (RSS) activity of P5 and its subcellular localization were determined in Nicotiana benthamiana plants by Agrobacterium tumefaciens-mediated expression assays and green fluorescent protein RNA silencing induction. Protein P5 partially suppressed local RNA silencing, strongly suppressed systemic RNA silencing and triggered reactive oxygen species accumulation. The P5 self-interacted and showed subcellular locations in plasmodesmata, endoplasmic reticulum and nucleus. Furthermore, P5 rescued the cell-to-cell movement of a movement defective mutant PVXΔP25 of potato virus X (PVX) and enhanced the pathogenicity of PVX. The N-terminal 1-89 amino acids of the P5 were responsible for the self-interaction ability and RSS activity, for which the signal peptide at positions 1-19 was indispensable. This study demonstrated the function of an emaravirus protein as a pathogenic factor suppressing plant RNA silencing to enhance virus infection and as an enhancer of virus movement.

Pear ring rot disease is an important branch disease, caused by Botryosphaeria dothidea. With the discovery of fungal viruses, the use of their attenuated properties for biological control provides a new strategy for the biological control of fungal disease. RNA silencing is a major antiviral defense mechanism in plants, insects, and fungi. Viruses encode and utilize RNA silencing suppressors to suppress host defenses. Previous studies revealed that Botryosphaeria dothidea chrysovirus 1 (BdCV1) exhibited weak pathogenicity and could activate host gene silencing by infecting B. dothidea. The aim of our study was to investigate whether BdCV1 can encode a silencing suppressor and what effect it has on the host. In this study, the capability of silencing inhibitory activity of four BdCV1-encoded proteins was analyzed, and the P3 protein was identified as a BdCV1 RNA silencing suppressor in the exotic host Nicotiana benthamiana line 16c. In addition, we demonstrated that P3 could inhibit local silencing, block systemic RNA silencing, and induce the necrosis reaction of tobacco leaves. Furthermore, overexpression of P3 could slow down the growth rate and reduce the pathogenicity of B. dothidea, and to some extent affect the expression level of RNA silencing components and virus-derived siRNAs (vsiRNAs). Combined with transcriptomic analysis, P3 had an effect on the gene expression and biological process of B. dothidea. The obtained results provide new theoretical information for further study of interaction between BdCV1 P3 as a potential silencing suppressor and B. dothidea.

Background Virus-induced gene silencing (VIGS) is a reverse genetics technology that can efficiently and rapidly identify plant gene functions. Although a variety of VIGS vectors have been successfully used in plants, only a few reports on VIGS technology in Luffa exist. Results In the present study, a new cucumber green mottle mosaic virus (CGMMV)-based VIGS vector, pV190, was applied to establish the CGMMV-VIGS to investigate the feasibility of the silencing system for Luffa . Phytoene desaturase ( PDS ) gene was initially selected as a VIGS marker gene to construct a recombinant vector. Plants infected with Agrobacterium harboring pV190- PDS successfully induced effective silencing in Luffa , and an effective gene silencing phenotype with obvious photobleaching was observed. To further validate the efficiency, we selected TEN for gene-silencing, which encodes a CYC/TB1-like transcription factor and is involved in tendril development. Luffa plants inoculated with the pV190- TEN exhibited shorter tendril length and nodal positions where tendrils appear are higher compared to those of non-inoculated plants. RT-qPCR showed that the expression levels of PDS and TEN were significantly reduced in the CGMMV-VIGS plants. Moreover, we evaluated the CGMMV-VIGS efficiency in three cucurbits, including cucumber, ridge gourd, and bottle gourd. Conclusion We successfully established a CGMMV-based VIGS system on ridge gourd and used marker genes to identify the feasibility of the silencing system in Luffa leaves and stems.

A 28-year-old woman presented to the ophthalmology department with a 3-year history of a tumor in her left eye. Examination revealed an isolated pink tumor in the conjunctiva with an associated large vessel (A). OCT angiography showed blood vessels in the shape of "coral" (B). After complete resection, pathological examination showed conjunctival papilloma (C). Conjunctival papilloma is an acquired benign tumor that originates from the conjunctival stratified squamous epithelium and usually progresses slowly. Most patients require surgical resection. Lesions may also be treated pharmacologically or via cryotherapy. (Magnified version of Figure A-C is available online at www.aaojournal.org).

Diseases caused by Alternaria alternata and Botryosphaeria dothidea diminish pear yield and quality, and restrict the pear agricultural industry. Lignification is a conserved mechanism for plant resistance against pathogen invasion. The regulatory mechanisms underlying defence-induced lignification in pear in response to fungal pathogen infection remain unknown. In this study, analysis of lignification level and lignin content in pear revealed that A. alternata and B. dothidea induced lignification, and transcriptomics showed that lignin biosynthesis was affected. To explore whether laccases (LACs) mediated by miR397 regulate lignification in pear, we investigated the role of PcmiR397 in repressing the expression of PcLACs using 5'-RNA ligase-mediated-RACE and co-transformation in tobacco. Opposite expression patterns for PcmiR397 and PcLAC target genes were observed in pear in response to pathogens. Transient transformation in pear demonstrated that silencing PcmiR397 and overexpressing a single PcLAC enhanced resistance to pathogens via lignin synthesis. To further reveal the mechanism underpinning the PcMIR397 response of pear to pathogens, the PcMIR397 promoter was analysed, and pMIR397-1039 was found to be inhibited by pathogen infection. The transcription factor PcMYB44 was up-regulated, and it bound to the PcMIR397 promoter and inhibited transcription following pathogen infection. The results demonstrate the role of PcmiR397-PcLACs in broad-spectrum resistance to fungal disease, and the potential role of PcMYB44 involved in the miR397-PcLAC module in regulating defence-induced lignification. The findings provide valuable candidate gene resources and guidance for molecular breeding to improve resistance to fungal disease in pear.

Many mycoviruses have been accurately and successfully identified in plant pathogenic fungus Botryosphaeria dothidea. This study discovered three mycoviruses from a B. dothidea strain SXD111 using high-throughput sequencing technology. A novel hypovirus was tentatively named Botryosphaeria dothidea hypovirus 1 (BdHV1/SXD111). The other two were known viruses, which we named Botryosphaeria dothidea polymycovirus 1 strain SXD111 (BdPmV1/SXD111) and Botryosphaeria dothidea partitivirus 1 strain SXD111 (BdPV1/SXD111). The genome of BdHV1/SXD111 is 11,128 nucleotides long, excluding the poly (A) tail. A papain-like cysteine protease (Pro), a UDP-glucose/sterol glucosyltransferase (UGT), an RNA-dependent RNA polyprotein (RdRp), and a helicase (Hel) were detected in the polyprotein of BdHV1/SXD111. Phylogenetic analysis showed that BdHV1/SXD111 was clustered with betahypovirus and separated from members of the other genera in the family Hypoviridae. The BdPmV1/SXD111 genome comprised five dsRNA segments with 2396, 2232, 1967, 1131, and 1060 bp lengths. Additionally, BdPV1/SXD111 harbored three dsRNA segments with 1823, 1623, and 557 bp lengths. Furthermore, the smallest dsRNA was a novel satellite component of BdPV1/SXD111. BdHV1/SXD111 could be transmitted through conidia and hyphae contact, whereas it likely has no apparent impact on the morphologies and virulence of the host fungus. Thus, this study is the first report of a betahypovirus isolated from the fungus B. dothidea. Importantly, our results significantly enhance the diversity of the B. dothidea viruses.