Targeting lymph nodes for enhanced cancer vaccination: From nanotechnology to tissue engineering.

Lymph nodes (LNs) occupy a critical position in initiating and augmenting immune responses, both spatially and functionally. In cancer immunotherapy, tumor-specific vaccines are blooming as a powerful tool to suppress the growth of existing tumors, as well as provide preventative efficacy against tumorigenesis. Delivering these vaccines more efficiently to LNs, where antigen-presenting cells (APCs) and T cells abundantly reside, is under extensive exploration. Formulating vaccines into nanomedicines, optimizing their physiochemical properties, and surface modification to specifically bind molecules expressed on LNs or APCs, are common routes and have brought encouraging outcomes. Alternatively, porous scaffolds can be engineered to attract APCs and provide an environment for them to mature, proliferate and migrate to LNs. A relatively new research direction is inducing the formation of LN-like organoids, which have shown positive relevance to tumor prognosis. Cutting-edge advances in these directions and discussions from a future perspective are given here, from which the up-to-date pattern of cancer vaccination will be drawn to hopefully provide basic guidance to future studies.

A vicinal oxygen chelate protein facilitates viral infection by triggering the unfolded protein response in Nicotiana benthamiana.

Vicinal oxygen chelate (VOC) proteins are members of an enzyme superfamily with dioxygenase or non-dioxygenase activities. However, the biological functions of VOC proteins in plants are poorly understood. Here, we show that a VOC in Nicotiana benthamiana (NbVOC1) facilitates viral infection. NbVOC1 was significantly induced by infection by beet necrotic yellow vein virus (BNYVV). Transient overexpression of NbVOC1 or its homolog from Beta vulgaris (BvVOC1) enhanced BNYVV infection in N. benthamiana, which required the nuclear localization of VOC1. Consistent with this result, overexpressing NbVOC1 facilitated BNYVV infection, whereas, knockdown and knockout of NbVOC1 inhibited BNYVV infection in transgenic N. benthamiana plants. NbVOC1 interacts with the basic leucine zipper transcription factors bZIP17/28, which enhances their self-interaction and DNA binding to the promoters of unfolded protein response (UPR)-related genes. We propose that bZIP17/28 directly binds to the NbVOC1 promoter and induces its transcription, forming a positive feedback loop to induce the UPR and facilitating BNYVV infection. Collectively, our results demonstrate that NbVOC1 positively regulates the UPR that enhances viral infection in plants.

Development of full-length infectious cDNA clones and host range identification of an echinacea strain of tobacco streak virus.

Tobacco streak virus induces severe diseases on a wide range of plants and becomes an emerging threat to crop yields. However, the infectious clones of TSV remain to be developed for reverse genetics studies. Here, we obtained the full genome sequence of a TSV-CNB isolate and analyzed the phylogenetic characteristics. Subsequently, we developed the full-length infectious cDNA clones of TSV-CNB driven by 35 S promoter using yeast homologous recombination. Furthermore, the host range of TSV-CNB isolate was determined by Agrobacterium infiltration and mechanical inoculation. The results reveal that TSV-CNB can infect 10 plant species in 5 families including Glycine max, Vigna radiate, Lactuca sativa var. Ramosa, Dahlia pinnate, E. purpurea, Calendula officinalis, Helianthus annuus, Nicotiana. Benthamiana, Nicotiana tabacum and Chenopodium quinoa. Taken together, the TSV infectious clones will be a useful tool for future studies on viral pathogenesis and host-virus interactions.

Predicting Prognosis and Immunotherapy Response in Multiple Cancers Based on the Association of PANoptosis-Related Genes with Tumor Heterogeneity.

PANoptosis is a newly recognized inflammatory pathway for programmed cell death (PCD). It participates in regulating the internal environment, homeostasis, and disease process in various complex ways and plays a crucial role in tumor development, but its mechanism of action is still unclear. In this study, we comprehensively analyzed the expression of 14 PANoptosis-related genes (PANRGs) in 28 types of tumors. Most PANRGs are upregulated in tumors, including Z-DNA binding protein 1 (ZBP1), nucleotide-binding oligomerization domain (NOD)-like receptor pyrin domain-containing 3 (NLRP3), caspase (CASP) 1, CASP6, CASP8, PYCARD, FADD, MAP3K7, RNF31, and RBCK1. PANRGs are highly expressed in GBM, LGG, and PAAD, while their levels in ACC are much lower than those in normal tissues. We found that both the CNV and SNV gene sets in BLCA are closely related to survival performance. Subsequently, we conducted clustering and LASSO analysis on each tumor and found that the inhibitory and the stimulating immune checkpoints positively correlate with ZBP1, NLRP3, CASP1, CASP8, and TNFAIP3. The immune infiltration results indicated that KIRC is associated with most infiltrating immune cells. According to the six tumor dryness indicators, PANRGs in LGG show the strongest tumor dryness but have a negative correlation with RNAss. In KIRC, LIHC, and TGCT, most PANRGs play an important role in tumor heterogeneity. Additionally, we analyzed the linear relationship between PANRGs and miRNA and found that MAP3K7 correlates to many miRNAs in most cancers. Finally, we predicted the possible drugs for targeted therapy of the cancers. These data greatly enhance our understanding of the components of cancer and may lead to the discovery of new biomarkers for predicting immunotherapy response and improving the prognosis of cancer patients.

The P1 protein of wheat yellow mosaic virus exerts RNA silencing suppression activity to facilitate virus infection in wheat plants.

Wheat yellow mosaic virus (WYMV) causes severe wheat viral disease in Asia. However, the viral suppressor of RNA silencing (VSR) encoded by WYMV has not been identified. Here, the P1 protein encoded by WYMV RNA2 was shown to suppress RNA silencing in Nicotiana benthamiana. Mutagenesis assays revealed that the alanine substitution mutant G175A of P1 abolished VSR activity and mutant Y10A VSR activity remained only in younger leaves. P1, but not G175A, interacted with gene silencing-related protein, N. benthamiana calmodulin-like protein (NbCaM), and calmodulin-binding transcription activator 3 (NbCAMTA3), and Y10A interacted with NbCAMTA3 only. Competitive Bimolecular fluorescence complementation and co-immunoprecipitation assays showed that the ability of P1 disturbing the interaction between NbCaM and NbCAMTA3 was stronger than Y10A, Y10A was stronger than G175A. In vitro transcript inoculation of infectious WYMV clones further demonstrated that VSR-defective mutants G175A and Y10A reduced WYMV infection in wheat (Triticum aestivum L.), G175A had a more significant effect on virus accumulation in upper leaves of wheat than Y10A. Moreover, RNA silencing, temperature, and autophagy have significant effects on the accumulation of P1 in N. benthamiana. Taken together, WYMV P1 acts as VSR by interfering with calmodulin-associated antiviral RNAi defense to facilitate virus infection in wheat, which has provided clear insights into the function of P1 in the process of WYMV infection.

High Expression of CDCA7 in the Prognosis of Glioma and Its Relationship with Ferroptosis and Immunity.

CDCA7 is a copy number amplification gene that promotes tumorigenesis. However, the clinical relevance and potential mechanisms of CDCA7 in glioma are unclear. CDCA7 expression level data were obtained from the Chinese Glioma Genome Atlas (CGGA) and The Cancer Genome Atlas (TCGA) databases, and the enriched genes and related signaling pathways were explored. Data on genes in CDCA7-related signaling pathways and nine marker genes of ferroptosis were retrieved and a protein-protein interaction (PPI) network analysis was performed. The correlation of CDCA7 to ferroptosis and tumor infiltration of 22 kinds of human immune cells and the association between CDCA7 and immune checkpoint molecules were analyzed. CDCA7 was significantly increased in gliomas in comparison to healthy tissues. Gene Ontology (GO) and gene set enrichment analysis (GSEA) revealed the impact of CDCA7 expression on multiple biological processes and signaling pathways. CDCA7 may affect ferroptosis by interacting with genes in the cell cycle pathway and P53 pathway. The increase in CDCA7 was positively correlated with multiple ferroptosis suppressor genes and genes involved in tumor-infiltrating immune cells and immune checkpoint molecules in glioma. CDCA7 can be a new prognostic factor for glioma, which is closely related to ferroptosis, tumor immune cell infiltration, and immune checkpoint.

Ferroptosis in colorectal cancer: Potential mechanisms and effective therapeutic targets.

Colorectal cancer (CRC) is a common malignant tumor characterized by unchecked division and survival of abnormal cells in the colon or rectum with high morbidity and mortality. Despite the rapid development of early screening methods and improved therapies, the prognosis of CRC is not satisfactory. Identification of new biomarkers for early detection and development of more effective therapies are still urgent tasks in current studies to achieve ideal treatment of CRC. Ferroptosis is a recently emerged novel regulated form of cell death characterized by a massive accumulation of iron-dependent lipid peroxidates, making it morphologically and molecularly distinct from apoptosis, cell death, and autophagy. Accumulating studies have shown that induction of ferroptosis in CRC successfully eliminates cancer cells resistant to other modes of cell death. Thus, ferroptosis may become a new direction for the design of CRC therapy. Although many research articles have investigated the possible roles of ferroptosis in CRC, a study that summarizes the main findings, including the regulators and mechanisms of action, of ferroptosis in CRC is not available. Herein, the studies in recent literature regarding the roles of ferroptosis on the progression and treatment of CRC were summarized, mainly focusing on molecular and biological mechanisms in vitro and in vivo. In particular, the roles of numerous ferroptosis regulators, such as SLC7A11, reactive oxygen species (ROS), glutathione (GSH), and iron, in CRC, were discussed and the application of ferroptosis-associated genes for the early diagnosis and prognosis of CRC was explored. In addition, an outlook for future studies of ferroptosis in CRC treatment and the possible barriers and the corresponding solutions were discussed.

First report of tobacco streak virus on Echinacea purpurea (Linn.) Moench in China.

Tobacco streak virus (TSV) is a member of the genus Ilarvirus in the family Bromoviridae (Vinodkumar et al. 2017). TSV is transmitted by thrips, seeds, pollen, and mechanical injury and has a broad host range, causing severe damage to several horticultural, oil and food crops including tobacco, sunflower, peanut, cotton, and soybean (Zambrana-Echevarría et al. 2021). TSV is now distributed mainly in the United States (McDaniel et al. 1992; Zambrana-Echevarría et al. 2021), India (Jain et al. 2008), Iran (Hosseini et al. 2012), Australia (Sharman et al. 2009) and Mexico (Silva-Rosales et al. 2013). Purple coneflower (Echinacea purpurea L.) is widely grown in China as an important herbal ornamental plant. In June 2020, Echinacea purpurea with the symptoms of necrosis lesions, malformation, and stunting were observed in the field of Haidian district, Beijing, China (40°2'69″ N, 116°28'28″ E) (Supplementary Fig. 1A). Total RNA of leaf tissue extracted using the hot borate method (Liang et al. 2020) was used for high-throughput sequencing on Illumina HiSeq X-10 platform at Biomarker Technologies (Beijing, China). Overall, 23,988,298 reads were generated. The final contigs assembled by Mega-Hit (v1.2.9) and Cap3 (Version Date: 02/10/15) were subjected to BLAST against GenBank using BLASTn and BLASTx algorithms. Of these contigs, 297 shared high nucleotide sequence similarities to the genomic sequence of broad bean wilt virus 2, while 9 contigs showed high nt sequence similarities (95-100%) to the genomic sequence of TSV. To confirm the presence of TSV, 30 randomly selected samples from Haidian district (40°2'69″ N, 116°28'28″ E) were tested by the double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) using a TSV specific monoclonal antibody (Agdia, SAR 25500/0500), where 18 samples were positive. In addition, total RNAs from 4 DAS-ELISA positive plants were extracted for TSV detection by reverse transcription-polymerase chain reactions (RT-PCR) using primer pair specific for the coat protein gene of TSV (TSV-CP-F, 5'-ATGAATACTTTGATCCAAGGTCC-3'; TSV-CP-R, 5'-TCAGTCTTGATTCACCAGAAAA-3'). The fragment with the expected size (~700 bp) was amplified in all 4 plants (Supplementary Fig. 1B) and subjected to direct Sanger sequencing. The CP gene of TSV CNB isolate was deposited in GenBank (MZ542767) and shared 100% sequence identity at the nucleotide level with the Gyp isolate infecting Ajuga reptans from Australia (JX463347.1). Furthermore, the local lesion host Chenopodium quinoa was used to purify and propagate TSV by mechanical inoculation with infected leaf sap. A pure culture of the TSV CNB isolate was obtained by single local lesion isolation after 3 serial passages on C. quinoa and back inoculated on E. purpurea seedlings. Systemic symptomology including leaf malformation was observed on E. purpurea three weeks post-inoculation (Supplementary Fig. 2A). The existence of TSV in two symptomatic leaf samples of E. purpurea was further verified by RT-PCR using specific primer pair (TSV-CP-F/R) (Supplementary Fig. 2B). In addition, the purified TSV CNB isolate was also inoculated to Nicotiana tabacum (Supplementary Fig. 2C). As previously reported (More et al. 2017), the Nicotiana tabacum plants infected with TSV developed typical streaks in systemic leaves. To the best of our knowledge, this is the first report of TSV on E. purpurea in China. This finding will assist further investigation into the epidemiology of diseases caused by TSV in China. Future studies are required to determine the incidence and impact that TSV might have on E. purpurea and other hosts in China.

Ethylene increases the cold tolerance of apple via the MdERF1B-MdCIbHLH1 regulatory module.

Cold stress has always been a major abiotic factor affecting the yield and quality of temperate fruit crops. Ethylene plays a critical regulatory role in the cold stress response, but the underlying molecular mechanisms remain elusive. Here, we revealed that ethylene positively modulates apple responses to cold stress. Treatment with 1-aminocyclopropane-1-carboxylate (an ethylene precursor) and aminoethoxyvinylglycine (an ethylene biosynthesis inhibitor) respectively increased and decreased the cold tolerance of apple seedlings. Consistent with the positive effects of ethylene on cold stress responses, a low-temperature treatment rapidly induced ethylene release and the expression of MdERF1B, which encodes an ethylene signaling activator, in apple seedlings. Overexpression of MdERF1B significantly increased the cold tolerance of apple plant materials (seedlings and calli) and Arabidopsis thaliana seedlings. A quantitative real-time PCR analysis indicated that MdERF1B upregulates the expression of the cold-responsive gene MdCBF1 in apple seedlings. Moreover, MdCIbHLH1, which functions upstream of CBF-dependent pathways, enhanced the binding of MdERF1B to target gene promoters as well as the consequent transcriptional activation. The stability of MdERF1B-MdCIbHLH1 was affected by cold stress and ethylene. Furthermore, MdERF1B interacted with the promoters of two genes critical for ethylene biosynthesis, MdACO1 and MdERF3. The resulting upregulated expression of these genes promoted ethylene production. However, the downregulated MdCIbHLH1 expression in MdERF1B-overexpressing apple calli significantly inhibited ethylene production. These findings imply that MdERF1B-MdCIbHLH1 is a potential regulatory module that integrates the cold and ethylene signaling pathways in apple.

Reaction coupling separation for isosteviol production from stevioside catalyzed by acidic ion-exchange resin.

Isosteviol, a prodrug used to be obtained via Wagner-Meerwein rearrangement from steviol with low yield and long reaction time. Herein, an in-situ separation-coupling-reaction is presented to prepare isosteviol from the natural sweetener stevioside. Simply with in-situ water-washing, the product containing 92.98% purity of isosteviol was obtained with a stevioside conversion of 97.23% from a packet bed reactor without further separation. Within the assayed inorganic acid, organic acids and acidic ionic liquids, the acidic ion-exchange resins provided higher product specificity towards isosteviol. Furthermore, comparing to 5-Fluorouracil, the product presented similar and even stronger inhibition on proliferation of the assayed human cancer cells in a time and dose-dependence by causing cell phase arrest. Isosteviol treatment caused G1 arrest on SGC-7901, HCT-8 and HCT-116 cells, S arrest on HepG2, Huh-7 and HepG3B cells, and G2 arrest on MGC-803 cells, respectively. Reaction coupling separation for isosteviol production catalyzed by acidic ion-exchange resin.

Antioxidant and hepatoprotective effects against acute CCl4 -induced liver damage in mice from red-fleshed apple flesh flavonoid extract.

Apple (Malus domestica Borkh.) is an important fruit tree species worldwide. Apple fruits are favored by consumers because of their antioxidative, anti-inflammatory, and antitumor effects as well as their protective effects against cardiovascular diseases and other health benefits. There is considerable interest in red-fleshed apple fruits among breeders because of their high flavonoid and anthocyanin contents. However, the flavonoids extracted from red-fleshed apple fruits must still be functionally characterized, especially regarding their protective effects against certain pathologies. In this study, the flavonoid components and contents in the extracts prepared from red-fleshed apple cultivar "Meihong" were determined. Additionally, the in vitro antioxidant activities and protective effects of the extracts against CCl4 -induced acute liver injury were investigated. The red-fleshed apple flesh flavonoid extract (RAFF) exhibited strong in vitro antioxidant activities. Compared with the model control mice treated with CCl4 , the mice pretreated with high (800 mg/kg·bw), middle (400 mg/kg·bw), and low (200 mg/kg·bw) RAFF doses had significantly lower CCl4 -induced serum aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase activities. Moreover, the RAFF pretreatment also significantly decreased the liver malondialdehyde activity and prevented the CCl4 -induced decrease in liver superoxide dismutase, glutathione peroxidase, catalase, and reduced glutathione levels. Furthermore, a histopathological examination revealed that RAFF inhibited the inflammatory cell infiltration and cell boundary loss caused by CCl4 in the liver. Thus, RAFF is a natural antioxidant with significant antioxidative activities and liver protective effects. The results of this study may be relevant for enhancing the application of the red-fleshed apple fruit extract as a food additive. PRACTICAL APPLICATION: We took the self-selected red-fleshed apple cultivar "Meihong" as the unique research material, and the active ingredients of its flavonoid extract, in vitro antioxidant activity and hepatoprotective effect were analyzed. It is of great significance to promote the development of the red-fleshed apple industry, and also provides an important reference for the development of natural antioxidants.

HCPro Suppression of Callose Deposition Contributes to Strain-Specific Resistance Against Potato Virus Y.

Potato virus Y (PVY; Potyviridae) is a continuing challenge for potato production owing to the increasing popularity of strain-specific resistant cultivars. Hypersensitive resistance (HR) is one type of plant defense responses to restrict virus spread. In many potato cultivars, such as cultivar Premier Russet (PR), local necrosis at the site of infection protects against the most common PVYO strain, but the HR often fails to restrain necrotic strains, which spread systemically. Here, we established the role of callose accumulation in the strain-specific resistance responses to PVY infection. We first uncovered that PVY, independent of the strain, is naturally capable of suppressing pathogenesis-related callose formation in a susceptible host. Such activity can be dissociated from viral replication by the transient expression of the viral-encoded helper component proteinase (HCPro) protein, identifying it as the pathogen elicitor. However, unlike the necrotic strain, PVYO and its corresponding HCPro are unable to block callose accumulation in resistant PR potatoes, in which we observed an abundance of callose deposition and the inability of the virus to spread. The substitution of eight amino acid residues within the HCPro C-terminal region that differ between PVYO and PVYN strains and were previously shown to be responsible for eliciting the HR response, are sufficient to restore the ability of HCProO to suppress callose accumulation, despite the resistant host background, in line with a new viral function in pathogenicity.

The B-box zinc finger protein MdBBX20 integrates anthocyanin accumulation in response to ultraviolet radiation and low temperature.

Ultraviolet-B (UV-B) radiation and low temperature promote the accumulation of anthocyanins, which give apple skins their red colour. Although many transcription regulators have been characterized in the UV-B and low-temperature pathways, their interregulation and synergistic effects are not well understood. Here, a B-box transcription factor gene, MdBBX20, was characterized in apple and identified to promote anthocyanin biosynthesis under UV-B conditions in field experiments and when overexpressed in transgenic apple calli. The transcript level of MdBBX20 was significantly induced by UV-B. Specific G-box elements in the promoters of target genes were identified as interaction sites for MdBBX20. Further experimental interrogation of the UV-B signalling pathways showed that MdBBX20 could interact with MdHY5 in vitro and in vivo and that this interaction was required to significantly enhance the promoter activity of MdMYB1. MdBBX20 also responded to low temperature (14°C) with the participation of MdbHLH3, which directly bound a low temperature-response cis elements in the MdBBX20 promoter. These findings demonstrate the molecular mechanism by which MdBBX20 integrates low-temperature- and UV-B-induced anthocyanin accumulation in apple skin.

Development of Beet necrotic yellow vein virus-based vectors for multiple-gene expression and guide RNA delivery in plant genome editing.

Many plant viruses with monopartite or bipartite genomes have been developed as efficient expression vectors of foreign recombinant proteins. Nonetheless, due to lack of multiple insertion sites in these plant viruses, it is still a big challenge to simultaneously express multiple foreign proteins in single cells. The genome of Beet necrotic yellow vein virus (BNYVV) offers an attractive system for expression of multiple foreign proteins owning to a multipartite genome composed of five positive-stranded RNAs. Here, we have established a BNYVV full-length infectious cDNA clone under the control of the Cauliflower mosaic virus 35S promoter. We further developed a set of BNYVV-based vectors that permit efficient expression of four recombinant proteins, including some large proteins with lengths up to 880 amino acids in the model plant Nicotiana benthamiana and native host sugar beet plants. These vectors can be used to investigate the subcellular co-localization of multiple proteins in leaf, root and stem tissues of systemically infected plants. Moreover, the BNYVV-based vectors were used to deliver NbPDS guide RNAs for genome editing in transgenic plants expressing Cas9, which induced a photobleached phenotype in systemically infected leaves. Collectively, the BNYVV-based vectors will facilitate genomic research and expression of multiple proteins, in sugar beet and related crop plants.

The ethylene response factor MdERF1B regulates anthocyanin and proanthocyanidin biosynthesis in apple.

KEY MESSAGE: The regulator MdERF1B in the apple (Malus × domestica) ethylene pathway mainly acts on MdMYB9 and MdMYB11 to regulate anthocyanin and proanthocyanidin accumulation. Dietary anthocyanins and proanthocyanidins (PAs) have health benefits for humans, and are associated with decreased risks of coronary heart disease and cancer. Ethylene can enhance reddening of apple (Malus × domestica), but the regulatory mechanism is poorly understood. In this study, an ethylene response factor (ERF), MdERF1B, was identified and functionally characterized. 'Orin' calli overexpressing MdERF1B were generated and then analyzed by quantitative reverse transcription-PCR. Compared with the control calli, the MdERF1B-overexpressing calli showed increased expression levels of MdACO1, MdERF1, and MdERF3 in the ethylene pathway and MdCHS, MdCHI, MdF3H, MdDFR, MdANS, MdLAR, MdANR, MdMYB9 and MdMYB11 in the flavonoid pathway. As a result, the levels of anthocyanins and PAs were significantly increased in the MdERF1B-overexpressing calli. MdERF1B interacted with MdMYB9, MdMYB1, and MdMYB11 proteins in yeast two-hybrid, pull-down, and bimolecular fluorescence complementation assays. Furthermore, in yeast one-hybrid and electrophoretic mobility shift assays, MdERF1B also bound to the promoters of MdMYB9, MdMYB1, and MdMYB11. In a luciferase reporter assay, MdERF1B mainly activated proMdMYB9 and proMdMYB11, promoting their expression levels. This was in agreement with MdERF1B's overexpression in calli, which barely affected MdMYB1 expression. Taken together, our findings provide an insight into the regulatory mechanisms in the ethylene pathway that increase anthocyanin and PA accumulation in apple.

The Conserved Proline18 in the Polerovirus P3a Is Important for Brassica Yellows Virus Systemic Infection.

ORF3a, a newly identified non-AUG-initiated ORF encoded by members of genera Polerovirus and Luteovirus, is required for long-distance movement in plants. However, the mechanism of action of P3a in viral systemic movement is still not clear. In this study, sequencing of a brassica yellows virus (BrYV) mutant defective in systemic infection revealed two-nucleotide variation at positions 3406 and 3467 in the genome. Subsequent nucleotide substitution analysis proved that only the non-synonymous substitution (C→U) at position 3406, resulting in P3aP18L, abolished the systemic infection of BrYV. Preliminary investigation showed that wild type BrYV was able to load into the petiole of the agroinfiltrated Nicotiana benthamiana leaves, whereas the mutant displayed very low efficiency. Further experiments revealed that the P3a and its mutant P3aP18L localized to the Golgi apparatus and near plasmodesmata, as well as the endoplasmic reticulum. Both P3a and P3aP18L were able to self-interact in vivo, however, the mutant P3aP18L seemed to form more stable dimer than wild type. More interestingly, we confirmed firstly that the ectopic expression of P3a of other poleroviruses and luteoviruses, as well as co-infection with Pea enation mosaic virus 2 (PEMV 2), restored the ability of systemic movement of BrYV P3a defective mutant, indicating that the P3a is functionally conserved in poleroviruses and luteoviruses and is redundant when BrYV co-infects with PEMV 2. These observations provide a novel insight into the conserved function of P3a and its underlying mechanism in the systemic infection.

Brassica yellows virus P0 protein impairs the antiviral activity of NbRAF2 in Nicotiana benthamiana.

In interactions between poleroviruses and their hosts, few cellular proteins have been identified that directly interact with the multifunctional virus P0 protein. To help explore the functions of P0, we identified a Brassica yellows virus genotype A (BrYV-A) P0BrA-interacting protein from Nicotiana benthamiana, Rubisco assembly factor 2 (NbRAF2), which localizes in the nucleus, cell periphery, chloroplasts, and stromules. We found that its C-terminal domain (amino acids 183-211) is required for self-interaction. A split ubiquitin membrane-bound yeast two-hybrid system and co-immunoprecipitation assays showed that NbRAF2 interacted with P0BrA, and co-localized in the nucleus and at the cell periphery. Interestingly, the nuclear pool of NbRAF2 decreased in the presence of P0BrA and during BrYV-A infection, and the P0BrA-mediated reduction of nuclear NbRAF2 required dual localization of NbRAF2 in the chloroplasts and nucleus. Tobacco rattle virus-based virus-induced gene silencing of NbRAF2 promoted BrYV-A infection in N. benthamiana, and the overexpression of nuclear NbRAF2 inhibited BrYV-A accumulation. Potato leafroll virus P0PL also interacted with NbRAF2 and decreased its nuclear accumulation, indicating that NbRAF2 may be a common target of poleroviruses. These results suggest that nuclear NbRAF2 possesses antiviral activity against BrYV-A infection, and that BrYV-A P0BrA interacts with NbRAF2 and alters its localization pattern to facilitate virus infection.

Rice black streaked dwarf virus P7-2 forms a SCF complex through binding to Oryza sativa SKP1-like proteins, and interacts with GID2 involved in the gibberellin pathway.

As a core subunit of the SCF complex that promotes protein degradation through the 26S proteasome, S-phase kinase-associated protein 1 (SKP1) plays important roles in multiple cellular processes in eukaryotes, including gibberellin (GA), jasmonate, ethylene, auxin and light responses. P7-2 encoded by Rice black streaked dwarf virus (RBSDV), a devastating viral pathogen that causes severe symptoms in infected plants, interacts with SKP1 from different plants. However, whether RBSDV P7-2 forms a SCF complex and targets host proteins is poorly understood. In this study, we conducted yeast two-hybrid assays to further explore the interactions between P7-2 and 25 type I Oryza sativa SKP1-like (OSK) proteins, and found that P7-2 interacted with eight OSK members with different binding affinity. Co-immunoprecipitation assay further confirmed the interaction of P7-2 with OSK1, OSK5 and OSK20. It was also shown that P7-2, together with OSK1 and O. sativa Cullin-1, was able to form the SCF complex. Moreover, yeast two-hybrid assays revealed that P7-2 interacted with gibberellin insensitive dwarf2 (GID2) from rice and maize plants, which is essential for regulating the GA signaling pathway. It was further demonstrated that the N-terminal region of P7-2 was necessary for the interaction with GID2. Overall, these results indicated that P7-2 functioned as a component of the SCF complex in rice, and interaction of P7-2 with GID2 implied possible roles of the GA signaling pathway during RBSDV infection.

The molecular mechanism underlying anthocyanin metabolism in apple using the MdMYB16 and MdbHLH33 genes.

KEY MESSAGE: MdMYB16 forms homodimers and directly inhibits anthocyanin synthesis via its C-terminal EAR repressor. It weakened the inhibitory effect of MdMYB16 on anthocyanin synthesis when overexpressing MdbHLH33 in callus overexpressing MdMYB16. MdMYB16 could interact with MdbHLH33. Anthocyanins are strong antioxidants that play a key role in the prevention of cardiovascular disease, cancer, and diabetes. The germplasm of Malus sieversii f. neidzwetzkyana is important for the study of anthocyanin metabolism. To date, only limited studies have examined the negative regulatory mechanisms underlying anthocyanin synthesis in apple. Here, we analyzed the relationship between anthocyanin levels and MdMYB16 expression in mature Red Crisp 1-5 apple (M. domestica) fruit, generated an evolutionary tree, and identified an EAR suppression sequence and a bHLH binding motif of the MdMYB16 protein using protein sequence analyses. Overexpression of MdMYB16 or MdMYB16 without bHLH binding sequence (LBSMdMYB16) in red-fleshed callus inhibited MdUFGT and MdANS expression and anthocyanin synthesis. However, overexpression of MdMYB16 without the EAR sequence (LESMdMYB16) in red-fleshed callus had no inhibitory effect on anthocyanin. The yeast one-hybrid assay showed that MdMYB16 and LESMdMYB16 interacted the promoters of MdANS and MdUFGT, respectively. Yeast two-hybrid, pull-down, and bimolecular fluorescence complementation assays showed that MdMYB16 formed homodimers and interacted with MdbHLH33, however, the LBSMdMYB16 could not interact with MdbHLH33. We overexpressed MdbHLH33 in callus overexpressing MdMYB16 and found that it weakened the inhibitory effect of MdMYB16 on anthocyanin synthesis. Together, these results suggested that MdMYB16 and MdbHLH33 may be important part of the regulatory network controlling the anthocyanin biosynthetic pathway.

MYB12 and MYB22 play essential roles in proanthocyanidin and flavonol synthesis in red-fleshed apple (Malus sieversii f. niedzwetzkyana).

Flavonoids are major polyphenol compounds in plant secondary metabolism. Wild red-fleshed apples (Malus sieversii f. niedzwetzkyana) are an excellent resource because of their much high flavonoid content than cultivated apples. In this work, R6R6, R6R1 and R1R1 genotypes were identified in an F1 segregating population of M. sieversii f. niedzwetzkyana. Significant differences in flavonoid composition and content were detected among the three genotypes by ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry analysis. Furthermore, two putative flavonoid-related genes encoding R2R3-MYB transcription factors, designated MYB12 and MYB22, were cloned and characterized. The expression patterns of MYB12 and MYB22 directly correlated with those of leucoanthocyanidin reductase and flavonol synthase, respectively. Their roles in flavonoid biosynthesis were identified by overexpression in apple callus and ectopic expression in Arabidopsis. MYB12 expression in the Arabidopsis TT2 mutant complemented its proanthocyanidin-deficient phenotype. Likewise, MYB22 expression in an Arabidopsis triple mutant complemented its flavonol-deficient phenotype. MYB12 could interact with bHLH3 and bHLH33 and played an essential role in proanthocyanidin synthesis. MYB22 was found to activate flavonol pathways by combining directly with the flavonol synthase promoter. Our findings provide a valuable perspective on flavonoid synthesis and provide a basis for breeding elite functional apples with a high flavonoid content.