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.

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.

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.

Chondrocyte is considered the only cell type in cartilage. However, the cell heterogeneity of chondrocytes in human articular cartilage is still not well defined, which hinders our understanding of the pathogenesis of osteoarthritis (OA). Here, we constructed a single-cell transcriptomic atlas of chondrocytes in healthy cartilage and identified nine chondrocyte subsets including homeostatic chondrocytes, proliferate fibrochondrocytes, and hypertrophic chondrocytes (HTC). Interestingly, we identified two distinct HTC subpopulations, among which HTC-1 specifically expressed genes associated with apoptosis and programmed cell death. We identified two main trajectories of chondrocytes, one of which differentiates into fibrochondrocytes, while the other terminates in apoptosis. Comparison of chondrocyte subsets between healthy and OA cartilage showed that proliferate fibrochondrocytes and HTC-1 expanded in OA patients, whereas homeostatic chondrocytes decreased. Interestingly, we discovered an OA-specific proliferate fibrochondrocyte subset that may contribute to the development of OA via inflammation. In summary, this study significantly enhanced our understanding of cell heterogeneity of chondrocytes in articular cartilage and provides insight into the pathogenesis of OA.

Immunosuppressive myeloid cells hinder immunotherapeutic efficacy in tumors, but the precise mechanisms remain undefined. Here, by performing single-cell RNA sequencing in colorectal cancer tissues, we found tumor-associated macrophages and granulocytic myeloid-derived suppressor cells increased most compared to their counterparts in normal tissue and displayed the highest immune-inhibitory signatures among all immunocytes. These cells exhibited significantly increased expression of immunoreceptor tyrosine-based inhibitory motif-bearing receptors, including SIRPA . Notably, Sirpa −/− mice were more resistant to tumor progression than wild-type mice. Moreover, Sirpα deficiency reprogramed the tumor microenvironment through expansion of TAM_ Ccl8 hi and gMDSC_ H2-Q10 hi subsets showing strong antitumor activity. Sirpa −/− macrophages presented strong phagocytosis and antigen presentation to enhance T cell activation and proliferation. Furthermore, Sirpa −/− macrophages facilitated T cell recruitment via Syk/Btk-dependent Ccl8 secretion. Therefore, Sirpα deficiency enhances innate and adaptive immune activation independent of expression of CD47 and Sirpα blockade could be a promising strategy to improve cancer immunotherapy efficacy.

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.

IntroductionEthylene (ET) is involved in plant responses to viral infection. However, its molecular mechanisms and regulatory network remain largely unknown.Methods and resultsIn the present study, we report that cucumber green mottle mosaic virus (CGMMV) in watermelon (Citrullus lanatus) triggers ET production by inducing the expression of ClACO5, a key gene of the ET biosynthesis pathway through transcriptome data analysis and gene function validation. The knock-down of ClACO5 expression through virus-induced gene silencing in watermelon and overexpressing ClACO5 in transgenic Nicotiana benthamiana indicated that ClACO5 positively regulates CGMMV resistance and ET biosynthesis. The salicylic acid-responsive transcription factor gene ClWRKY70 shares a similar expression pattern with ClACO5. We demonstrate that ClWRKY70 directly binds to the W-box cis-element in the ClACO5 promoter and enhances its transcription. In addition, ClWRKY70 enhances plant responses to CGMMV infection by regulating ClACO5 expression in watermelon.DiscussionOur results demonstrate that the ClWRKY70-ClACO5 module positively regulates resistance to CGMMV infection in watermelon, shedding new light on the molecular basis of ET accumulation in watermelon in response to CGMMV infection.

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.

Anti-PD-1 therapy has shown promising outcomes in the treatment of different types of cancer. It is of fundamental interest to analyze the efficacy of anti-PD-1 therapy in cancer patients infected with hepatitis B virus (HBV) since the comorbidity of HBV and cancer is widely documented. We designed a multicenter retrospective study to evaluate the efficacy of anti-PD-1 therapy on non-liver cancer patients infected with HBV. We found anti-PD-1 therapy achieved much better outcomes in HBV+ non-liver cancer patients than their HBV- counterparts. We performed single-cell RNA sequencing (scRNA-seq) on peripheral blood mononuclear cells (PBMCs) from esophageal squamous cell carcinoma (ESCC) patients. We found both cytotoxicity score of T cells and MHC score of B cells significantly increased after anti-PD-1 therapy in HBV+ ESCC patients. We also identified CX3CR1high TEFF, a subset of CD8+ TEFF, associated with better clinical outcome in HBV+ ESCC patients. Lastly, we found CD8+ TEFF from HBV+ ESCC patients showing higher fraction of Exhaustionhi T than their HBV- counterpart. In summary, anti-PD-1 therapy on HBV+ non-liver cancer patients is safe and achieves better outcomes than that on HBV- non-liver cancer patients, potentially because HBV+ patients had higher fraction of Exhaustionhi T, which made them more efficiently respond to anti-PD-1 therapy.

Pear ring rot disease,caused by the pathogenic agent of Botryosphaeria dothidea.Botryosphaeria dothidea chrysovirus 1(BdCV1)induced to the hypovirulence of B.dothidea LW-C and LW-1,which will be used as an ideal candidate material for biological control of fruit tree ring rot disease.In order to further clarify whether the BdCV1-mediated hypovirulent strain has potential as a good candidate biocontrol resource,LW-C and LW-1 was confrontly cultured with the B.dothidea strains isolated and collected from different geographical origins and Valsa pyri strain XJ-28,which caused serious harm to pear trees,respectively.The obtained derivate strains were detected to be positive for BdCV1,whose growth rates and pathogenicity were further determined and analyzed.The results showed that BdCV1 could be successfully horizontally transmitted to different B.dothidea and V pyri strains with certain frequencies,which further inhibited the growth rates and induced to hypovirulence of the strains.The obtained results revealed BdCV1 could be successfully transmitted to the recipient strains at a certain frequency,which could cause the growth rate to decrease and inhibit the growth of the recipient strains to different extent.The formed lesion extension lengths of derivate strains with BdCV1 range from about two to four times longer than that of the recipient strains,when they are inoculated with pear branches or fruits.It further demonstrated that BdCV1 can inhibit the virulence of recipient strains.In summary,BdCV1 could be horizontally transmitted to different recipient strains,revealing that it has wide host range.It is predicted that BdCV1 could be used as a potential biocontrol factor in pear fungal disease biocontrol in practice.

AbstractRNA silencing, a core part of plants' antiviral defence, requires the ARGONAUTE, DICER‐like, and RNA‐dependent RNA polymerase proteins. However, how these proteins contribute to watermelon's RNA interference (RNAi) pathway response to cucumber green mottle mosaic virus (CGMMV) has not been characterized. Here, we identify seven ClAGO, four ClDCL, and 11 ClRDR genes in watermelon and analyse their expression profiles when infected with CGMMV. ClAGO1 and ClAGO5 expression levels were highly induced by CGMMV infection. The results of ClAGO1 and ClAGO5 overexpression and silencing experiments suggest that these genes play central roles in watermelon's antiviral defence. Furthermore, co‐immunoprecipitation and bimolecular fluorescence complementation experiments showed that ClAGO1 interacts with ClAGO5 in vivo, suggesting that ClAGO1 and ClAGO5 co‐regulate watermelon defence against CGMMV infection. We also identified the ethylene response factor (ERF) binding site in the promoters of the ClAGO1 and ClAGO5 genes, and ethylene (ETH) treatment significantly increased ClAGO5 expression. Two ERF genes (Cla97C08G147180 and Cla97C06G122830) closely related to ClAGO5 expression were identified using co‐expression analysis. Subcellular localization revealed that two ERFs and ClAGO5 predominantly localize at the nucleus, suggesting that enhancement of resistance to CGMMV by ETH is probably achieved through ClAGO5 but not ClAGO1. Our findings reveal aspects of the mechanisms underlying RNA silencing in watermelon against CGMMV.

Understanding the mechanisms underlying plant resistance to virus infections is crucial for viral disease management in agriculture. However, the defense mechanism of watermelon (Citrullus lanatus) against cucumber green mottle mosaic virus (CGMMV) infection remains largely unknown. In this study, we performed transcriptomic, metabolomic, and phytohormone analyses of a CGMMV susceptible watermelon cultivar 'Zhengkang No.2' ('ZK') and a CGMMV resistant wild watermelon accession PI 220778 (PI) to identify the key regulatory genes, metabolites, and phytohormones responsible for CGMMV resistance. We then tested several phytohormones and metabolites for their roles in watermelon CGMMV resistance via foliar application, followed by CGMMV inoculation. Several phenylpropanoid metabolism-associated genes and metabolites, especially those involved in the flavonoid biosynthesis pathway, were found to be significantly enriched in the CGMMV-infected PI plants compared with the CGMMV-infected 'ZK' plants. We also identified a gene encoding UDP-glycosyltransferase (UGT) that is involved in kaempferol-3-O-sophoroside biosynthesis and controls disease resistance, as well as plant height. Additionally, salicylic acid (SA) biogenesis increased in the CGMMV-infected 'ZK' plants, resulting in the activation of a downstream signaling cascade. SA levels in the tested watermelon plants correlated with that of total flavonoids, and SA pre-treatment up-regulated the expression of flavonoid biosynthesis genes, thus increasing the total flavonoid content. Furthermore, application of exogenous SA or flavonoids extracted from watermelon leaves suppressed CGMMV infection. In summary, our study demonstrates the role of SA-induced flavonoid biosynthesis in plant development and CGMMV resistance, which could be used to breed for CGMMV resistance in watermelon.

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.