Both small heat shock protein and J-Domain protein direct defense against Areca palm velarivirus 1 (APV1) by degrading coat protein via autophagy pathway

Both small heat shock protein and J-Domain protein direct defense against Areca palm velarivirus 1 (APV1) by degrading coat protein via autophagy pathway | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Both small heat shock protein and J-Domain protein direct defense against Areca palm velarivirus 1 (APV1) by degrading coat protein via autophagy pathway Xi Huang, Xianmei Cao, Jie Lu, Zengyu Xing, Jingling Zhai, Hongxing Wang, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5886236/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Both autophagy and heat shock proteins (HSPs) play dual roles in promoting or inhibiting viral infections. However, the coordination between autophagy and HSPs in the defense against viral infections remains underexplored, and the underlying mechanisms are still poorly understood. This study first revealed an interaction between a cytosolic small heat shock protein (AcsHSP) and a type II J-domain protein (AcDNAJB13) of areca palm with the coat protein (CP) of Areca Palm Velarivirus 1 (APV1) and the interaction is independent of the HSP70 chaperones. The closest homologs in Nicotiana benthamiana (NbsHSP and NbDNAJB13) also interacted with CP. Both AcsHSP and AcDNAJB13 were localized in the cytoplasm and nucleus, and co-expression with CP altered AcsHSP intracellular localization. APV1 infection or transient CP expression induced the expression of AcsHSP and AcDNAJB13 , which, in turn, inhibited CP accumulation. Virus-induced gene silencing (VIGS) of NbsHSP and NbDNAJB13 significantly increased the accumulation of transiently expressed CP-GFP. CP degradation occurred via an autophagic pathway. Both AcsHSP and AcDNAJB13 interacting with AcATG8f1, and these interactions were required for CP degradation. Furthermore, silencing endogenous NbsHSP and NbDNAJB13 enhanced APV1 replication, while overexpression of AcsHSP reduced APV1 accumulation. Our findings demonstrate that AcsHSP and AcDNAJB13 function as selective cargo receptors for CP degradation via autophagy pathway, thereby limiting APV1 infection and offering new insights into the roles of heat shock protein families. Biological sciences/Microbiology/Virology/Viral pathogenesis Biological sciences/Plant sciences/Plant immunity/Effectors in plant pathology Areca palm velarivirus 1 autophagy small heat shock protein J-domain protein cargo receptor Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Autophagy is an evolutionarily conserved cellular mechanism responsible for degrading and recycling damaged or unnecessary intracellular components, essential for maintaining cellular homeostasis 1 . A key feature of autophagy is the formation of double-membrane vesicles known as autophagosomes 1, 2, 3 . These structures originate from the phagophore assembly site (PAS), which primarily derives from the endoplasmic reticulum (ER) 4 . The maturation of autophagosomes from the PAS is regulated by a series of autophagy-related genes (ATGs) 5 , of which, ATG8 is critical for phagophore expansion and closure. ATG8 is conjugated to the membrane lipid phosphatidylethanolamine (PE), a process known as ATG8 lipidation. This lipidation is a hallmark of autophagosome formation and aids in cargo selection 6 . The precursor ATG8 is initially cleaved by the protease ATG4 to expose a C-terminal glycine, enabling subsequent activation by the ATP-dependent enzyme ATG7. This activation facilitates its transfer to ATG3, after which ATG8 is covalently linked to PE by the ATG5-ATG12-ATG16 hexametric complex, which functions as a ligase 7, 8 . ATG8 interacts with several proteins (such as ATG1, Beclin1/ATG6, and ATG7) that are essential for cargo recruitment and autophagosome signaling. Once fully formed, the autophagosome, containing the sequestered damaged organelles and cellular debris, fuses with the vacuole, where its contents are degraded and recycled 9, 10, 11 . Emerging findings highlight the pivotal role of autophagy in plant virus infections 1, 12, 13, 14 . Initially documented by Haxim et al. (2017), autophagy functions as an antiviral mechanism targeting and degrading the βC1 protein of plant geminiviruses through interaction with ATG8 15 . Hafren et al. (2017) demonstrated that the P4 protein of cauliflower mosaic virus (CaMV) interacts with the autophagy receptor NEIGHBOR OF BRCA1 (NBR1), leading to selective degradation 16 . Turnip mosaic virus (TuMV) encoded NIb protein interacts directly with Beclin1/ATG6, utilizing it as a selective autophagy receptor for NIb degradation 17 . Conversely, some plant viruses suppress or co-opt autophagy to facilitate their infection. For instance, BaMV infection induces the expression of ATGs, utilizing autophagy to selectively engulf viral RNA-containing chloroplasts, aiding viral replication or evading host defenses 18 . The viral F-box protein P0 induces autophagy to degrade membrane-bound ARGONAUTE1 (AGO1) 4 . Barley stripe mosaic virus (BSMV) γb disrupts ATG7-ATG8 interaction, suppressing autophagosome formation and promoting viral infection 19 . VPg of TuMV facilitates the degradation of suppressor of gene silencing 3 (SGS3) by autophagy and ubiquitination 20 . In summary, autophagy exhibits both antiviral and proviral dual roles during plant viral infections, reflecting its complex interplay with viral pathogenesis. Heat shock proteins (HSPs) are a group of molecular chaperones that respond to stress conditions. They play crucial roles in preventing protein aggregation, and promoting correct protein folding 21, 22 . HSPs vary widely in molecular weight, ranging from approximately 10 to 100 kDa, and are classified into several families including small heat shock proteins (sHSPs), HSP40 (DNAJ or J-Domain proteins), HSP60, HSP70, HSP90, and large heat shock proteins 23, 24 . sHSPs are characterized as 12–25 kDa polypeptides containing the α-crystallin domain (ACD), which shares homology with the α-crystallins found in the eye lens 25, 26, 27 . In angiosperms, eleven classes of sHSP genes have been identified 26, 27 . J-domain proteins could be classified into four types (I-IV). Type Ⅰ proteins contain a J-domain with a tripeptide HPD motif for interaction with HSP70, in addition, both the zinc-finger motif and the C-terminal domain which are involved in substrate binding, while type Ⅱ retains only the latter and type Ⅲ comprises only the J-domain. Type IV only has a “J-like proteins”, which lack the HPD motif in the J-domain 28 . While HSP family members play roles in promoting or inhibiting viral infections in animal cells, they generally support viral infection in plants 29, 30 . Movement protein of Rice stripe virus (RSV) and Tomato mosaic virus (ToMV) hijack DNAJA proteins (NbMIP1s) to protect itself from autophagic degradation 31 . HSPs interact with viral replicase complexes (VRCs) and other viral macromolecular protein complexes, aiding in viral processes such as replication, translocation, assembly, disassembly, and movement during plant RNA virus infections 24, 32, 33, 34 . In summary, both autophagy and heat shock proteins (HSPs) exhibit dual roles as proviral and antiviral factors in viral infections. However, the coordination between autophagy and HSPs in defending against viral infections is poorly documented, and the underlying mechanisms remain poorly understood. Areca palm velarivirus 1 (APV1) is a member of the genus Velarivirus in the family Closteroviridae , identified in samples from yellowing leaf disease (YLD) affecting Areca catechu 35, 36 . APV1 is transmitted by the mealybugs, causing yellow leaf symptoms on Areca catechu 37, 38, 39 . APV1 infection leads to the disassembly of chloroplasts, and the resultant leaf yellowing may serve as a signal for transmission vectors 40 . However, the molecular mechanisms underlying these processes remain largely unknown. To investigate the interaction between APV1 and its plant host, a Yeast Two-Hybrid assay (Y2H) was used to screen for proteins that interact with the coat protein (CP) of APV1, leading to the identification of a sHSP and a DNAJ protein. Further studies revealed that both HSPs function as selective cargo receptors for autophagic degradation of APV1 CP. This represents the first example of both sHSP and J-Domain protein playing an antiviral role via autophagy pathway. Results Small heat shock protein (AcsHSP) and J-domain protein (AcDNAJB13) interact with APV1 CP To investigate potential cellular factors that may interact with APV1, we conducted a yeast two-hybrid (Y2H) screening to identify proteins binding to the APV1 coat protein (APV1-CP). This screening identified ten host candidate proteins that specifically interact with CP (Table S1). Among these, we selected a small heat shock protein, AcsHSP (18.0 kDa), and a J-domain protein, AcDNAJB13 (37.9 kDa), for further investigation. AcsHSP features a 16-amino-acid C-terminal extension (CTE), a typical α-crystallin domain (ACD) spanning amino acids 50-141, and a 49-amino-acid N-terminal domain (NTD) characterized by a CⅠ NTD motif, “IFDPFSLDVWDPF” (9-21aa) (Fig. S1A and B). AcsHSP was categorized as a cytosolic class I (CI) protein based on the classification method 27 . AcDNAJB13 contains a J-domain (3-81 aa) featuring the highly conserved tripeptide HPD, which facilitates binding to the ATPase domain of HSP70. Additionally, it has a C-terminal domain (173-336 aa) for substrate binding but lacks a Zn-finger domain (Fig. S1A and C), classifying AcDNAJB13 as a member of the type II J-domain protein family 28 . The interaction between AcsHSP-CP and AcDNAJB13-CP was further confirmed through pairwise Y2H assays, which detected strong interactions between AcDNAJB13 and CP, as well as between AcsHSP and CP (Fig. 1A). To validate these interactions in planta , we conducted bimolecular fluorescence complementation (BiFC) experiments. The host genes AcsHSP and AcDNAJB13 were individually fused to the N-terminal part of YFP, generating nYFP-AcsHSP and nYFP-AcDNAJB13, respectively, while CP was fused to the C-terminal part of YFP to create cYFP-CP. Positive interactions between cYFP-CP and nYFP-AcsHSP, as well as between cYFP-CP and nYFP-AcDNAJB13, were confirmed by the presence of YFP fluorescence, indicating that the two HSPs interact with CP in vivo (Fig. 1B). Glutathione S-transferase (GST) Pull-down assays confirmed that both GST-AcsHSP and GST-AcDNAJB13 could capture His-tagged CP, demonstrating that both HSPs interact with CP in vitro (Fig. 1C). These findings were further supported by co-immunoprecipitation (Co-IP) assays, which demonstrated that Myc-AcsHSP and Myc-AcDNAJB13 individually co-precipitated with GFP-CP (Fig. 1D and E). Collectively, these assays confirm that both AcsHSP and AcDNAJB13 interact with CP in vivo and in vitro . To identify which domain of AcDNAJB13 is involved in its interaction with CP, we constructed truncated versions of AcDNAJB13 based on the predicted domains and tested them. BiFC assays revealed that the 1–81 aa and 1–172 aa fragments interacted with CP, whereas the 82–345 aa and 173–345 fragments did not. The interaction pattern of the 1–81 aa fragment was similar to those of the full-length AcDNAJB13 (Fig. S2), demonstrating that the J-domain of AcDNAJB13 interacts with CP. Furthermore, both AcsHSP and AcDNAJB13 interacted with AcHSP70 (Fig.S3A), deletion of the HPD tripeptide from the J-domain (AcDnaJB13-∆HPD) abolished the interaction with AcHSP70, suggesting that the HPD motif is essential for interaction between the AcDNAJB13 and HSP70 (Fig. S3B). However, AcDnaJB13-∆HPD still interacted with CP in the BiFC assay (Fig. S3C), indicating that the interaction between AcDnaJB13 and CP is independent of the HSP70 chaperone. We have built an infectious cDNA clone of APV1 which established systemic infection in inoculated N. benthamiana via agroinoculation, indicating that N. benthamiana is a compatible host of APV1 (unpublished data). Closest homologs of AcsHSP and AcDNAJB13 in N. benthamiana were found by BLAST searches against the SGN database, then cloned and designated hereafter as NbsHSP and NbDNAJB13 , respectively. BiFC experiments confirmed the interactions of NbsHSP-CP and NbDNAJB13-CP in vivo (Fig. S4), implying similar roles of NbsHSP and NbDNAJB13 in response to APV1 infection. CP alters the subcellular localization of AcsHSP Viral infections often disturbs the subcellular distribution of host proteins, which affects the plant-virus interaction 41 . Therefore, we examined the intracellular distribution of CP, AcsHSP and AcDNAJB13 to determine whether such changes of subcellular localization occur for AcsHSP and AcDNAJB13 in response to CP expression. First, the CP-GFP fusion protein (N-terminal GFP tag) was transiently expressed in N. benthamiana . Confocal microscopy showed that CP-GFP was localized in both the cytoplasm and the nucleus in epidermal cells (Fig. 2A). Next, the two HSPs were transiently expressed alone or co-expressed with CP, respectively. Similar to the circumstance of CP, fluorescence of AcsHSP-GFP or AcDNAJB13-GFP alone was detected in both the cytoplasm and the nucleus (Fig. 2B and 2C). Interestingly, when AcsHSP-GFP was transiently expressed alone, multiple punctate fluorescence spots appeared in the cytoplasm. Such punctate spots were disappeared when CP were transiently co-expressed. Apparently, CP altered the intracellular localization of AcsHSP, preventing AcsHSP from forming punctate structures (Fig. 2B). In similar co-expression assays of AcDNAJB13 and CP, the presence of CP seemed to have no effect on the cellular distribution of AcDNAJB13 (Fig. 2C). RFP-tagged AcsHSP or AcDNAJB13 was transiently co-expressed with CP-GFP, overlapped fluorescence was detected in both the cytoplasm and the nucleus, confirmed their interaction and co-localization (Fig. 2B and 2C). APV1 infection induces the expression of AcsHSP and AcDNAJB13 , which conversely impede CP accumulation To explore the roles of AcsHSP and AcDNAJB13 in APV1 infection, we used qRT-PCR to analyze changes of their expression patterns in mock-treated and APV1-affected plants. As closteroviruses are known to take a long time (usually two months and longer) to establish systemic infection after inoculation 38, 39 , areca seedlings were inoculated with APV1 via mealybug transmission and sampled at 5 months post-inoculation (mpi), while N. benthamiana plants were inoculated with a full-length infectious cDNA clone of APV1 via agroinoculation (unpublished data) and sampled at 3 mpi. The results indicated that AcsHSP and AcDNAJB13 in areca seedlings, as well as their counterparts NbsHSP and NbDNAJB13 in N. benthamiana , were all upregulated under systemic infection of APV1 (Fig. 3A-B). Further, Flag-CP transiently expressed in N. benthamiana enhanced the transcription of NbsHSP and NbDNAJB13 (Fig. 3C-D) , suggesting that expression of CP alone is capable to induce the expression of both HSPs. To assess the effect of induced AcsHSP and AcDNAJB13 expression on CP, the protein accumulation levels of CP-GFP were examined in N. benthamiana with or without transiently expressed HSPs. Immunoblotting analysis revealed that the levels of CP-GFP decreased when co-expressed with Myc-AcsHSP (Fig. 3E), as well as when co-expressed with Myc-AcDNAJB13 (Fig. 3F). On the contrary, when a TRV-based virus-induced gene silencing (VIGS) vector was employed to silence NbsHSP and NbDNAJB13, the accumulation level of CP-GFP remarkedly increased, while inoculation of TRV-GUS (served as control) just slightly improved CP accumulation (Fig. 3G and 3H). These results indicate that both AcsHSP and AcDNAJB13 play a key role in CP degradation. Degradation of CP is independent of the UPR pathway Infecting virus hijacks translation machinery to produce large amounts of viral proteins, which inevitably perturbs ER homeostasis and often causes unfolded protein response (UPR) 42 . Inspired by that a type-A J-domain proteins NbMIP1s induced by UPR protects RSV protein from autophagic degradation 31 , we investigated whether APV1 infection can induce the UPR. RT-qPCR was applied to analyze the expression of the marker genes for the UPR and ER stress. NbbZIP60, NbbZIP17-1, NbPDI, NbSKP1-1, NbSKP1-4 , and NbbZIP17-2/3 were significantly induced in APV1 infected N. benthamiana, AcbZIP17, AcbZIP60 and AcbZIP28 were induced in APV1 infected areca palm (Fig S5A), indicating that APV1 infection induces the UPR in plant cells. We further studied whether the degradation of CP is UPR dependent. Previous reports have shown that infiltration of 2mM dithiothreitol (DTT) into N. benthamiana leaves could efficiently trigger ER stress, activate the UPR and autophagy pathway 31 . However, DTT induced UPR and autophagy did not affect the CP stability (Fig S5B), suggesting that degradation of CP is independent of the UPR pathway. APV1 CP is targeted for autophagic degradation To explore the mechanisms involved in the degradation of CP, we assessed its stability in vivo by treating N. benthamiana leaves expressing CP-GFP with cycloheximide (CHX), a known inhibitor of protein synthesis. Following CHX treatment, the synthesis of CP is halted, yet its degradation continues. Immunoblotting revealed a notable decrease in CP-GFP levels within 4 hours of CHX treatment, while free GFP remained stable throughout the observation period (Fig. 4A), indicating that CP is susceptible to degradation within the plant. In eukaryotes, protein degradation primarily occurs through two pathways: the ubiquitin-proteasome and autophagy pathways. To elucidate the degradation pathway of APV1-CP, we employed specific chemical inhibitors targeting both pathways. Leaves expressing APV1-GFP were treated with E64D and 3-MA (inhibitors of autophagy) and MG132 (an inhibitor of the 26S proteasome). Western blot analysis demonstrated that CP-GFP accumulation increased significantly upon treatment with E64D and 3-MA, whereas MG132 treatment did not affect CP levels (Fig. 4B), indicating that CP is primarily degraded via the autophagy pathway. To investigate whether CP is delivered to autophagosomes, we utilized RFP-tagged AcATG8f1-1 and NbATG8f1 as markers for autophagosomes, examining the localization of CP-GFP. Confocal microscopy showed that CP-GFP colocalized with both RFP-AcATG8f1-1 and RFP-NbATG8f1, forming distinct punctate structures resembling autophagic bodies. These punctate structures were absent in the control group co-expressing CP-GFP with free RFP (Fig. 4C). To further validate the autophagic degradation of APV1 CP, we silenced key genes involved in cargo recognition and autophagosome signaling using TRV-mediated virus-induced gene silencing (VIGS). Silencing of ATG3A, ATG5, Beclin1, and ATG8f1 significantly enhanced the stability of CP-GFP during transient expression (Fig. 5A). We subsequently monitored CP degradation rates in vivo by treating leaves expressing CP-GFP with CHX and collecting samples at 0 and 6 hours post-treatment (hpt). While TRV-GUS inoculation (control) led to a moderate increase in CP accumulation, co-expression with TRV constructs targeting NbATG3, NbATG5, NbATG7, NbBeclin1, or NbATG8 resulted in significantly elevated levels of CP-GFP at 6 hpt (Fig. 5B). The silencing efficiency of these genes was confirmed through qRT-PCR (Fig. 5C). Collectively, these findings demonstrate that CP is transported to autophagosomes and undergoes degradation via the canonical autophagy pathway. Autophagy is induced by APV1 infection or CP expression To investigate whether APV1 infection triggers autophagy, we conducted RT-PCR to analyze the expression of autophagic marker genes following APV1 infection. The results showed that all tested marker genes ( AcATG8c-1, AcATG8c-2, AcATG8f, AcATG3A1, AcATG3B1, AcATG5A, and AcATG7A ) were significantly upregulated in APV1-infected areca palms. In contrast, NbATG3, NbATG5, and NbATG8c were induced by APV1 in N. benthamiana (Fig. 6A-B). Consistent with these findings, Western blot analysis revealed an accumulation of NbATG8-II, a marker of activated autophagy, in samples infected with APV1 (Fig. 6C). To further assess autophagy induction by APV1, we employed transmission electron microscopy (TEM) to monitor autophagic activity. The APV1 infection resulted in a significantly higher number of double-membrane structures characteristic of autophagosomes in the cytoplasm compared to mock-inoculated controls, indicating an enhanced level of autophagic flux in APV1-infected N. benthamiana (Fig. 6D). We also examined whether autophagy was induced by the CP; transient expression of a Flag-tagged CP in N. benthamiana led to a strong induction of NbATG8-II, as shown by Western blot analysis, compared to the mock inoculation with an empty vector (Fig. 6E). In summary, these results confirm that autophagy is induced by the CP following APV1 infection. However, it remains unclear whether other APV1-encoded proteins also contribute to this induction. Both AcsHSP and AcDNAJB13 interact with AcATG8f1 To determine whether AcsHSP and AcDNAJB13 directly mediate the degradation of CP through the autophagy pathway, we employed the Luciferase Complementation Assay (LCA) to examine the interaction between AcATG8f1 and both AcsHSP and AcDNAJB13. Results demonstrated that both AcsHSP and AcDNAJB13 interacted with AcATG8f1 in planta , exhibiting strong luciferase activity (Fig. 7A). These interactions were further validated by BiFC assay, which showed that the AcATG8f1-AcsHSP complex dispersed throughout the cytoplasm as abundant irregular granules, while the AcATG8f1-AcDNAJB13 complex predominantly localized to the cytomembrane and nucleus (Fig. 7B). This strongly suggests that AcsHSP and AcDNAJB13 play distinct roles in the autophagy pathway. To identify which domain of AcDNAJB13 interacts with AcATG8f1, we conducted interaction assays using truncated versions of AcDNAJB13. BiFC results indicated that the J-domain (1-81 aa) of AcDNAJB13 interacts with AcATG8f1, with extended peptides (1-171 aa) exhibiting stronger fluorescence. In contrast, the fragment spanning 82-345 aa showed no interaction (Fig. S6), suggesting that the region between 82-172 aa does not directly interact with AcATG8f1 but may regulate or enhance the interaction between the J-domain of AcDNAJB13 and AcATG8f1. These findings indicate that both AcsHSP and AcDNAJB13 may server as cargo receptors for CP degradation in the autophagy pathway. The interactions between CP and both AcsHSP and AcDNAJB13 are required for CP degradation To further determine whether the interactions between CP and both AcsHSP and AcDNAJB13 are necessary for CP degradation, BiFC assay was first used to identify which truncated fragment of CP interacts with AcsHSP and AcDNAJB13. BiFC revealed that the 1-110 aa fragment of CP interacted with both AcsHSP and AcDNAJB13, whereas the 111-205aa and 205-298aa fragments did not (Fig. S7A and S7B). Then, the truncated Frag-CP (111-298aa) was transiently co-expressed with myc-AcsHSP and myc-AcDNAJB13 in N. benthamiana , respectively. Seedlings were treated with CHX at 48 h post agroinfiltration. Samples were collected at 12 hours post-treatment (hpt). Western-blot indicated that the stability of truncated CP was not affected by expression of myc-AcsHSP (Fig. S7C) or myc-AcDNAJB13 (Fig. S7D) , indicating that the interactions between CP and both AcsHSP and AcDNAJB13 are required for CP degradation. Silencing NbsHSP or NbDNAJB13 promotes APV1 infection and over expressing AcsHSP inhibits APV1 infection. To determine whether the expression levels of NbsHSP or NbDNAJB13 influence APV1 infection, TRV-induced VIGS was used to silence NbsHSP or NbDNAJB13, then after, a GFP-labeled infectious clone for APV1 was constructed and agroinfiltrated into N. benthamiana to monitor the APV1 infection. TRV-triggered Silencing either NbsHSP or NbDNAJB13 significantly reduced the expression levels of NbsHSP or NbDNAJB13 (Fig. 8C) and promoted APV1 infection indicating by the GFP fluorescence intensity (Fig. 8A) and density (Fig. 8B), which was further verified by the Western blotting using antibody against GFP (Fig. 8D and 8E). In contrast, overexpressing ACsHSP in N. benthamiana substantially inhibited APV1 infection (Fig. 8F-H). These results strongly suggest that both sHSP and DNAJB13 play a role in the antiviral defense against APV1 infection through CP degradation via the autophagy pathway. Discussion Heat shock proteins (HSPs) exhibit both antiviral and proviral functions in viral infections of humans and animals 23, 43 . While there is limited documentation of their antiviral functions in plant, and the underlying mechanisms remain largely unexplored 29, 30 . This study presents novel findings on the roles of both sHSP and J-Domain proteins in mediating defense against plant virus through an autophagic degradation pathway. Based on protein structure predictions, 18.0 kD AcsHSP is categorized as a CI class sHSP, confirmed to localize in both the cytoplasm and the nucleus. CI sHSPs, along with HSP101, have been shown to protect eukaryotic translation initiation factors during heat stress (McLoughlin et al., 2016). However, there has yet to be any report of CI sHSPs participating in viral infections. Notably, the 23.6 kD sHSP from Triticum aestivum has been found to interact with the coat protein of the wheat yellow mosaic virus 44 , while the 22.8 kD sHSP from Citrullus lanatus interacts with the helicase of cucumber green mottle mosaic virus (CGMMV). Overexpression of this sHSP was associated with reduced viral RNA accumulation and slowed disease progression, while silencing the sHSP did not significantly affect CGMMV infection 41 , and the underlying mechanism remain unexplored. AcsHSP is the first example of sHSP playing an antiviral role via autophagy pathway. DNAJ/Hsp40 proteins are well-established as having both antiviral and proviral functions during infections caused by human viruses (Multhoff, 2006). For instance, the type I J-domain protein DNAJA3 has been shown to interact with the VP1 protein of the foot-and-mouth disease virus (FMDV), leading to its degradation through the autophagy pathway 45 . Conversely, the type II J-domain protein Hdj1 interacts with the core protein of hepatitis B virus, inhibiting viral replication by destabilizing viral proteins 46 . Another type II J-domain protein, DnaJB1, facilitates the replication of influenza A virus by aiding the nuclear import of viral ribonucleoproteins 47 . Type III J-domain protein DNAJC14 has been found to inhibit yellow fever virus and hepatitis C virus during the viral RNA replication phase 48 . In plant, type I J-domain proteins NbMIP1s interact with both the viral movement protein and the resistance protein Tm-2 2 . Their silencing compromises the stability of both proteins, exhibiting a dual functionality in both the resistance and the facilitation to virus infection 49 . The NbMIP1s interacts with RSV NSvc4, preventing its autophagic degradation and thus promoting viral infection 31 . A type III J-domain protein GmHSP40 identified in soybean plays a positive role in viral defense; its overexpression induces hypersensitive response-like cell death, while its silencing increases susceptibility to Soybean mosaic virus 50 . These findings highlight the complex roles of J-domain proteins in the ongoing conflict between viruses and plant hosts. This study focuses on the type II J-domain protein AcDNAJB13, which interacts with both viral CP and ATG8f, indicating that AcDNAJB13 is a selective cargo receptor for the degradation of CP through the autophagy pathway. AcsHSP and AcDNAJB13 do not possess any shared functional domains or sequence homology, prompting an intriguing inquiry into their similar roles in defense against APV1. Specifically, both HSPs have been shown to interact with CP (Fig. 1 and Fig. S4). Moreover, their respective gene expressions are upregulated following APV1 infection as well as the expression of CP (Fig. 3A-B). Additionally, both AcsHSP and AcDNAJB13 interact with ATG8f (Fig. 7) and are crucial for the autophagic degradation of CP (Fig. 3C-D). The closest homologs found in N. benthamiana , namely NbsHSP and NbDNAJB13, demonstrate a similar defensive role against APV1, suggesting that these two types of HSPs are conserved across the plant kingdom. Nevertheless, different functions between AcsHSP and AcDNAJB13 could be reflected by their cellular localization. On the one hand, the subcellular localization of AcsHSP was altered by CP appearance, while AcDNAJB13 was not (Fig. 2). On the other hand, BiFC revealed AcATG8f1-AcsHSP complex dispersed throughout the cytoplasm as abundant irregular granules, while the AcATG8f1-AcDNAJB13 complex predominantly localized to the cytomembrane and nucleus (Fig. 7B). These results indicate the distinct roles of AcsHSP and AcDNAJB13 playing in the plant cell, it requires further investigation. Autophagy can act as an antiviral mechanism by directly targeting viral proteins for degradation through ATG8, as seen with βC1 of geminiviruses 51 , C1 proteins from tomato leaf curl Yunnan virus (TLCYnV), and various other geminiviruses 12, 52 . In another autophagy pathway, cargo receptor is required to assist ATG8 in capturing viral proteins for degradation. For instance, NBR1 serves as a cargo receptor that targets the capsid protein P4 of Cauliflower mosaic virus (CaMV) for autophagic degradation 16 . NbP3IP directs the autophagic degradation of RSV p3 by interacting with both p3 and NbATG8f 53 . Virus-induced small peptide 1 (VISP1) acts as a cargo receptor exhibiting antiviral activity by mediating the autophagic degradation of various viral proteins, including CMV 2b, the P14 protein of pothos latent virus (PoLV), the C2 protein of beet severe curly top virus (BSCTV), and the AC2 protein of cabbage leaf curl virus (CaLCuV) 54, 55 . This work represents the first example that both sHSP and J-Domain protein function as selective autophagosomal cargo receptors for the autophagic degradation of viral protein, restricting virus infection. In summary, we propose a model illustrating the roles of AcsHSP and AcDNAJB13 in the APV1 infection cycle (Fig. 9). APV1 infection activates autophagy to target CP for degradation. APV1 also triggers the ER stress and the UPR. However, the degradation of CP is independent on UPR. The CP is captured intracellularly by both AcsHSP and AcDNAJB13, which is further recruited to the phagophore through ATG8's interactions with AcsHSP and AcDNAJB13. AcsHSP and AcDNAJB13 function as cargo receptors for autophagy. This work highlights the novel roles of sHSP and J-domain protein, offering new insights into antiviral function of HSP families. Methods Plant Materials and Virus Inoculation Areca palm seedlings were inoculated with mealybugs ( Ferrisia virgata or Pseudococcus cryptus ) carrying APV1, following previously established protocols 38, 39 . Nicotiana benthamiana was inoculated using an infectious cDNA clone of APV1 through agroinoculation. Transgenic lines of N. benthamiana expressing the coat protein (CP) of APV1 are maintained in our laboratory. All plants were cultivated in a growth chamber under controlled conditions of 25°C with a 16-hour light period and 18°C with an 8-hour dark period, maintaining a relative humidity of 75%. Subcellular Localization AcsHSP, AcDNAJB13, and CP were amplified via RT-PCR and cloned into the pCambia1302-GFP vector, resulting in constructs pCambia1302-GFP-AcsHSP, pCambia1302-GFP-AcDNAJB13, and pCambia1302-GFP-CP. Additionally, the coding sequences of AcsHSP and AcDNAJB13 were fused with the RFP protein to create constructs 35S::RFP-AcsHSP and 35S::RFP-AcDNAJB13. The vectors were infiltrated into the leaves of N. benthamiana , and fluorescence was observed using a Zeiss LSM 780 confocal microscope at 72 hours post-inoculation (hpi). All primers used in this study are listed in Table S2. Gene amplification was performed using PrimeSTAR® GXL DNA Polymerase (TaKaRa, Dalian), and all vectors were constructed using the ClonExpress® Ultra One Step Cloning Kit (Vazyme, Nanjing, China, Cat #C115). Yeast Two-Hybrid (Y2H) Screening To identify proteins that interact with APV1 CP, a Y2H cDNA library from areca palm was constructed by OE Biotech (Shanghai, China). The bait vector pGBKT7-CP was created, and Y2H screening was conducted using the Yeastmaker™ Yeast Transformation System 2 (Clontech, USA). To confirm the interaction candidates in yeast, the prey vectors pGADT7-AcsHSP and pGADT7-AcDNAJB13 were co-transformed with the bait vector pGBKT7-CP into the yeast strain Y187. The transformed yeast was plated on minimal media with triple dropouts (TDO, SD/-Trp/-Leu/-His) and quadruple dropouts (QDO, SD/-Trp/-Leu/-His/-Ade) supplemented with AbA and X-α-Gal, respectively. pGBKT7-53 and pGADT7-T served as positive controls, while pGBKT7-Lam and pGADT7-T served as negative controls. Three independent experiments were conducted to validate the results. Bimolecular Fluorescence Complementation (BiFC) and Complementation Luciferase Assay (CLA) in Planta The BiFC assay was conducted as previously described 31 . Vectors pFGC-YN173-AcsHSP, pFGC-YN173-AcDNAJB13, and pFGC-CN155-CP were created. The YN-CN combinations of vectors were agroinfiltrated into N. benthamiana leaves, and epidermal cells from 1-cm infiltrated leaf explants were collected at 72 hpi for observation using a Zeiss LSM 780 confocal microscope. CLA assays were performed as previously outlined 56 , with constructs AcDNAJB13-cLUC, AcsHSP-cLUC, and AcATG8f1-nLUC. The nLUC/cLUC vector combinations were co-infiltrated into N. benthamiana leaves through agroinoculation. Leaves were harvested at 48 hpi, treated with 1 mM luciferin, and imaged using the NightShade LB 985 In Vivo Plant Imaging System (Berthold Technologies, Germany) two minutes after luciferin treatment. In Vitro GST Pull-Down Assay The GST pull-down assay was performed according to previously established methods 41 . The coding sequences of AcsHSP and AcDNAJB13 were cloned into the pGEX-6p vector, while the open reading frame (ORF) of CP was cloned into the pET28a vector. GST-AcsHSP, GST-AcDNAJB13, and His-CP were expressed in E. coli BL21 (DE3) and purified using a GST-tag and His-tag Protein Purification Kit (Beyotime, Shanghai, China, Cat #P2226). The pull-down assay was conducted using the BersinBio™ GST-pulldown Kit (Cat #Bes3012) following the manufacturer's protocol. For each assay, 2 μg of His-CP protein was incubated with equal amounts of GST-AcDNAJB13, GST-AcsHSP, and GST on BersinBio GSH magnetic beads at 4°C for 12 hours. Proteins bound to the beads were collected, separated by SDS-PAGE, and analyzed by Western blot using anti-GST (TransGen Biotech, Beijing, China, Cat #HT601) and anti-His antibodies (TransGen Biotech, Beijing, China, Cat #HT501). In Vivo Co-Immunoprecipitation (Co-IP) Assay For Co-IP assays, plasmids pCambia1302-GFP-CP in combination with pCambia1302-Myc-AcHsp or pCambia1302-Myc-AcDNAJB13 were agroinfiltrated into N. benthamiana . The infiltrated leaves were harvested at 72 hpi, and total proteins were extracted from frozen, ground tissue using ice-cold extraction buffer (50 mM HEPES, pH 7.5, 150 mM KCl, 1 mM EDTA, and 0.5% Triton X-100), supplemented with 1 mM dithiothreitol and a 1× protease inhibitor cocktail (Roche) 53 . After centrifugation at 12,000 g for 10 minutes at 4°C, the supernatant was collected and incubated with Anti-GFP Magnetic Beads (Yamei, Shanghai, China, Cat #YJ010) at room temperature for one hour with gentle agitation. The precipitates were washed three times with IP buffer and analyzed by Western blotting using anti-Myc antibodies. Virus-Induced Gene Silencing (VIGS) Tobacco rattle virus (TRV)-based vectors pTRV2 containing partial gene fragments of NbsHSP, NbsDNAJB13, NbATG3A, NbATG5, NbBeclin1, NbATG7A, NbATG8f1 , or GUS were constructed and introduced into Agrobacterium tumefaciens strain GV3101. Agrobacterium cultures carrying TRV1 or TRV2-derived vectors were mixed in infiltration buffer (10 mM MgCl₂, 10 mM MES, and 200 mM acetosyringone) and infiltrated into N. benthamiana. The infiltrated plants were used for further analysis at 10 days post-inoculation (dpi). Western Blotting Total proteins from plant leaf samples were extracted using lysis buffer (50 mM Tris-HCl, pH 6.8, 4.5% SDS, 7.5% β-mercaptoethanol, 9 M urea). After centrifugation at 10,000 × g for 15 minutes at 4°C, the supernatant was collected for Western blot assays. Proteins were detected using monoclonal antibodies against GFP (1:5000; HT801; TransGen, Beijing, China), His (1:5000; HT501; TransGen), GST (1:5000; HT601; TransGen), and Flag (1:5000; R24091; ZEN-BIOSCIENCE, Chengdu, China), as well as anti-Myc (1:5000; 341173; ZEN-BIOSCIENCE). Monoclonal antibodies served as primary antibodies, while horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (1:5000; HS201; TransGen) or HRP-conjugated goat anti-rabbit IgG (1:5000; AS014; ABclonal, Wuhan, China) were used as secondary antibodies. Blots were visualized with a chemiluminescence film (Thermo Fisher SuperSignal West Pico PLUS Chemiluminescent Substrate, catalog no. 34577). Chemical Treatment Chemical treatments were performed as previously described 31 . Inhibitors of protein degradation pathways, including MG132 (cat: HY-13259, MCE, Monmouth Junction, USA), 3-MA (cat: HY-19312, MCE), and E64d (cat: HY-100229, MCE), were dissolved in DMSO and diluted in ddH₂O to final concentrations of 100 μM, 5 mM, and 100 μM, respectively. An equal volume of DMSO diluted in ddH₂O served as the control. CHX (cat: HY-12320, MCE) and DTT were directly dissolved in ddH₂O to final concentrations of 100 μM and 2 mM, respectively. After infiltration, leaves were kept moist for 12 hours and then harvested for Western blotting. Confocal Microscopy zonfocal imaging was conducted as described previously 57 . The eGFP fluorophore was excited at 488 nm with emission detected at 490-540 nm; the YFP fluorophore was excited at 514 nm with emission detected at 520-560 nm; and the RFP fluorophore was excited at 561 nm with emission detected at 560-620 nm. Images were captured using a laser scanning confocal microscope (LSM 780, Carl Zeiss) and processed with LSM software (Zen 2012, Carl Zeiss). Transmission Electron Microscopy (TEM) TEM was performed as previously described 40 . Briefly, betel palm leaves were cut into 2 mm × 2 mm pieces and fixed overnight at 4°C in a fixation buffer containing 2.5% glutaraldehyde and 0.05 M phosphate (pH 7.2). Samples were washed three times with fixation buffer and post-fixed in 2% OsO₄ at 4°C for two hours. Following dehydration through a graded ethanol series (50%, 70%, 80%, 90%, 95%, and 100%), tissue samples were embedded in Spurr’s resin. Ultrathin sections (70–90 nm) were prepared using a Leica EM UC7 ultramicrotome and stained sequentially with uranyl acetate for 20 minutes and Reynolds’ lead citrate for 5 minutes. Sections were observed using a Hitachi H-7650 or a JEM-1230 transmission electron microscope operating at 80 kV. Quantitative Reverse Transcription PCR (qRT-PCR) Total RNA was isolated using the Tiangen plant RNA isolation kit (DP441, Tiangen Biotech, Beijing, China). Reverse transcription of total RNA was performed using the Thermo Scientific RevertAid First Strand cDNA Synthesis Kit (K1622, Thermo Fisher Scientific, USA). qRT-PCR was conducted using TransStart Tip Green qPCR SuperMix (AQ141, TransGen Biotech, China). AcActin and NbActin were used as internal controls for areca and N. benthamiana , respectively. All experiments were performed with at least three biological replicates, and relative gene expression levels were calculated using the 2-ΔΔCt method. Primer sets used are listed in Table S2. Statistical Analysis All experiments were conducted at least three times unless otherwise specified. Statistical differences between samples were analyzed using a two-tailed, unpaired Student’s t-test with equal variance (computed in Microsoft Excel). Differences were considered statistically significant at P < 0.05, indicated in figure legends as *, P < 0.05 or **, P < 0.01. Declarations Accession numbers Gene sequences from this study can be found in GenBank under the following accession numbers: AcsHSP , PQ623096; AcDNAJB13 , PQ623095; AcATG8f1 ; NbsHSP , PQ623098; NbDNAJB13 , PQ623097; NbATG3A, KX369396; NbATG5, KX369397; NbBeclin1 , AY701316; NbATG7A , KX369398; NbATG8f1 ; MG733106. Acknowledgements This work was supported by grants from National Natural Science Foundation of China (Grants Nr. 32460408), Hainan Key Research and Development Plan Foundation (ZDYF2024XDNY208) and the earmarked fund for Agriculture Research System in Hainan Province (Grant No. HNARS-1-G4-1). Author contributions X.H. and R.Z. designed all experiments. X.C., J.L., Z.X., and H.W. performed the protein interaction experiments, virus inoculation, and blotting experiments. X.C. and L.J. performed the experiments for immunofluorescence staining and electron microscopy. H.W. and J.Z. analyzed the data. X.H. and R.Z. organized the project and wrote the manuscript. All authors read and approved the manuscript. Competing interests: The authors declare no competing interests. Data availability All data that support the findings of this study are available from the corresponding author upon request. Source data are provided with this paper. References Yang M, Liu Y. Autophagy in plant viral infection. FEBS Lett 596 , 2152-2162 (2022). Verchot J. The ER quality control and ER associated degradation machineries are vital for viral pathogenesis. Frontiers in plant science 5 , 66 (2014). Howell SH. Endoplasmic reticulum stress responses in plants. Annual review of plant biology 64 , 477-499 (2013). Michaeli S , et al. The viral F-box protein P0 induces an ER-derived autophagy degradation pathway for the clearance of membrane-bound AGO1. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5886236","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":418692970,"identity":"00b63e65-e575-4df5-8fbe-262fc7fcdacb","order_by":0,"name":"Xi Huang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8ElEQVRIiWNgGAWjYBACg8M8YFrOgIGxASqWQJwWY+K1SDZAtCRuQIgR0MLPzntM4ueO2vTt7IfbHvzccRgokmPA8HMHbi1szHxpkr1njufu7ElsN+w9c5hBsueNAWPvGXxaeMwkeNuO5W44kNgGZBxmMLiRY8DM2IbHYUAtkn/bjqUbnH/YBmQcZrAnpEWymcdMmretJsHgRmKbNNgWCQJagIFsbC3bdsBww42HbdKybek8EmeeFRzsxafl/BnDm2/b6uQNzqc/k3zbZi3H35688cFPPFqg4DCcBY6mAwQ1MDDUEaFmFIyCUTAKRiwAAMKfUGCchLHXAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-3605-2692","institution":"Hainan University","correspondingAuthor":true,"prefix":"","firstName":"Xi","middleName":"","lastName":"Huang","suffix":""},{"id":418692971,"identity":"ca4fc95b-e748-4f5b-8f46-cf04160006b6","order_by":1,"name":"Xianmei Cao","email":"","orcid":"","institution":"Hainan University","correspondingAuthor":false,"prefix":"","firstName":"Xianmei","middleName":"","lastName":"Cao","suffix":""},{"id":418692972,"identity":"1ace4667-6099-4a6a-a0ae-37c4c3f0675c","order_by":2,"name":"Jie Lu","email":"","orcid":"","institution":"Hainan University","correspondingAuthor":false,"prefix":"","firstName":"Jie","middleName":"","lastName":"Lu","suffix":""},{"id":418692973,"identity":"d608db75-b3a5-445b-84e0-fca79ef4d411","order_by":3,"name":"Zengyu Xing","email":"","orcid":"","institution":"Hainan University","correspondingAuthor":false,"prefix":"","firstName":"Zengyu","middleName":"","lastName":"Xing","suffix":""},{"id":418692974,"identity":"a414f6b5-cd2c-4edb-9574-294577ad2114","order_by":4,"name":"Jingling Zhai","email":"","orcid":"","institution":"Hainan University","correspondingAuthor":false,"prefix":"","firstName":"Jingling","middleName":"","lastName":"Zhai","suffix":""},{"id":418692975,"identity":"8fc11323-d905-4452-bd9a-5b707df81fa0","order_by":5,"name":"Hongxing Wang","email":"","orcid":"","institution":"Hainan University","correspondingAuthor":false,"prefix":"","firstName":"Hongxing","middleName":"","lastName":"Wang","suffix":""},{"id":418692976,"identity":"11f68fd1-d1b2-43e2-8365-1787d2d604ad","order_by":6,"name":"Ruibai Zhao","email":"","orcid":"","institution":"Hainan University","correspondingAuthor":false,"prefix":"","firstName":"Ruibai","middleName":"","lastName":"Zhao","suffix":""}],"badges":[],"createdAt":"2025-01-23 07:55:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5886236/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5886236/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":77232958,"identity":"d9c4e304-d069-45f7-a202-2f8df9635ff0","added_by":"auto","created_at":"2025-02-26 12:39:10","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":366601,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInteraction between CP and AcsHSP or AcDNAJB13. \u003c/strong\u003e(A) Yeast two-hybrid\u003cstrong\u003e \u003c/strong\u003edemonstrating interaction between CP and AcsHSP or AcDNAJB13. pGBK-53+pGAD-T combination is a positive control, and pGBK-Lam+pGAD-T is negative controls. The samples were plated onto minimal media triple dropouts (TDO, SD/-Trp/-Leu/-His) and quadruple dropouts (QDO, SD/-Trp/-Leu/-His/-Ade) with AbA and X-α-Gal, respectively. (B) Bimolecular fluorescence complementation (BiFC) assay demonstrating interaction between CP and AcsHSP or AcDNAJB13\u003cstrong\u003e \u003c/strong\u003ein the leaves of \u003cem\u003eN. benthamiana\u003c/em\u003e at 72 h post-infiltration (hpi). The N- or C-terminal fragments of YFP were fused to the C-terminus of AcsHSP or AcDNAJB13 and CP. Bars, 50 μm. (C) GST pull-down assay for detection of \u003cem\u003ein vitro\u003c/em\u003e interaction of CP with AcsHSP or AcDNAJB13. GST-AcsHSP or GST-AcDNAJB13 immobilized on glutathione-Sepharose beads was incubated with \u003cem\u003eE. coli\u003c/em\u003e-expressed recombinant CP-6×His protein. Beads were washed and proteins were analyzed by Western-blot assays using anti-His (upper panel) antibodies and anti-GST (lower panel), respectively. (D-E) CP co-immunoprecipitated (co-IP) with AcsHSP or AcDNAJB13. CP-GFP was co-expressed with AcsHSP-Myc (D) or AcDNAJB13-Myc (E) respectively in \u003cem\u003eN. benthamiana \u003c/em\u003eleaves through agroinfiltration. Coexpression of AcsHSP-Myc and GFP or Coexpression of AcDNAJB13-Myc and GFP served as a negative control. At 2 dpi, leaf lysates were immunoprecipitated with anti-GFP beads, then the immunoprecipitates were assessed by Western-blot assays using anti-Myc.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-5886236/v1/783a1c688b7d4f46fc617421.png"},{"id":77233476,"identity":"6facc345-fe59-4c16-b1e1-db7faa5b3a86","added_by":"auto","created_at":"2025-02-26 12:47:10","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":426255,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSubcellular localization of CP, AcsHSP and AcDNAJB13.\u003c/strong\u003e (A) CP-GFP fusion protein was transiently expressed in \u003cem\u003eN. benthamiana\u003c/em\u003e. Gw5-RFP and Ghd7-RFP were co-expressed as cell membrane marker and nucleus marker respectively. (B, C) Localization of AcsHSP-GFP and AcDNAJB13-GFP in leaves of CP-OE transgenic \u003cem\u003eN. benthamiana \u003c/em\u003ewas first imaged by confocal microscopy. Arrows indicate the scattered punctate distribution of sHSP-GFP fluorescence. Then, AcsHSP-RFP or AcDNAJB13-RFP was agroinfiltrated with CP-GFP in leaves of \u003cem\u003eN. benthamiana\u003c/em\u003e. The leaves were observed by confocal microscope at 60 hpi. Bars=50 μm. Gw5-RFP and Ghd7-RFP were co-expressed respectively.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-5886236/v1/e3946d93ce64c1b99cd9a46b.png"},{"id":77232960,"identity":"66052a96-c0e0-4a19-889a-ed96f3148ed8","added_by":"auto","created_at":"2025-02-26 12:39:10","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":344381,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInduction of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003esHSP\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eDnaJB13\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003etriggers the degradation of CP.\u003c/strong\u003e (A-B) APV1 infection induced expression of \u003cem\u003esHSP\u003c/em\u003e and \u003cem\u003eDnaJB13\u003c/em\u003e\u003cem\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003eqRT-PCR analysis of the transcript levels of \u003cem\u003eAcsHSP\u003c/em\u003e and \u003cem\u003eAcDnaJB13\u003c/em\u003e in Areca palm 5 months post APV1 inoculation (mpi) and of closest homologies \u003cem\u003eNbsHSP\u003c/em\u003e and \u003cem\u003eNbDnaJB13 \u003c/em\u003ein \u003cem\u003eN. benthamiana \u003c/em\u003eat 3mpi. The housekeeping gene \u003cem\u003eActin\u003c/em\u003e served as the internal control. The data are given as a relative ratio of gene expression compared to a control (mock) set at 1.0. The error bars reflect the standard error of the mean (n=3). The asterisks indicate a significant difference as determined by the Student’s \u003cem\u003et\u003c/em\u003etest (*P 0.05 or **P 0.01). (C) Transient expression Flag-CP induced \u003cem\u003eNbsHSP\u003c/em\u003eand \u003cem\u003eNbDnaJB13 \u003c/em\u003ein \u003cem\u003eN. benthamiana\u003c/em\u003e. (D) Western-blot imagines the Flag-CP expression in \u003cem\u003eN. benthamiana \u003c/em\u003eat 2 days post agroinoculation using anti-Flag antibody, empty vector containing Flag-label was used as negative control. (E-F) CP-GFP was co-expressed with Myc-AcsHSP (E) or with Myc-AcDNAJB13 in \u003cem\u003eN. benthamiana \u003c/em\u003e(F). Western blotting was conducted 2 days post agroinoculation using anti-GFP (upper panel) and anti-Myc (lower panel) antibodies. (G-H) TRV-based virus-induced gene silencing (VIGS) vectors with \u003cem\u003eNbsHSP\u003c/em\u003e(118-302bp coding sequence) and\u003cem\u003e NbDNAJB13 \u003c/em\u003e(1-300bp coding sequence)were constructed and agroinfiltrated into \u003cem\u003eN. benthamiana \u003c/em\u003ewhich transiently expressing CP-GFP. Western-blot was applied to analyze the CP-GFP accumulation using anti-GFP antibody 72 hpi. TRV-GUS was used as control.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-5886236/v1/b6ed1355043af90a5e9f63de.png"},{"id":77232979,"identity":"2458a067-644a-4a4c-aa2c-54fd5af3b249","added_by":"auto","created_at":"2025-02-26 12:39:11","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":258056,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAPV1 CP is unstable in plant cells and degradation of CP is blocked by inhibitors of autophagy. (\u003c/strong\u003eA) \u003cem\u003eIn vivo\u003c/em\u003e protein stability assay of APV1 CP. The leaves transientlyexpressing CP-GFP were treated with 100 μM CHX. Four infiltrated leaves were individually collected for Western-blotting from each treatment at 0h and 4h post inoculation. (B) Inhibitors treatment of \u003cem\u003eN. benthamiana\u003c/em\u003e expressing CP-GFP. The leaves expressing CP-GFP were treated with 100 μM CHX together with MG132, E64d, 3MA, or DMSO, respectively. Six hours after treatment, the leaves were harvested for total protein extraction and western blotting. (C) Confocal images of CP-GFP co-expressed with RFP-NbATG8f1. \u003cem\u003eN. benthamiana\u003c/em\u003e leaves were infiltrated with agrobacteria carrying CP-GFP and RFP-NbATG8f1 or RFP. Bars, 50 μm.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-5886236/v1/d8f0a02c444fc7d80365cfd2.png"},{"id":77233479,"identity":"faa5b8ec-13d1-4d6e-ab21-0b387c812d16","added_by":"auto","created_at":"2025-02-26 12:47:11","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":297074,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTRV-triggered silencing autophagy components enhances the stability of CP.\u003c/strong\u003e (A) CP-GFP was co-expressed with TRV-GUS, TRV-ATG3A, TRV-ATG5, TRV-Beclin1, TRV-ATG7A or TRV-ATG8f1 in \u003cem\u003eN\u003c/em\u003e. \u003cem\u003ebenthamiana\u003c/em\u003e. Leaves samples were collected at 72 hpi for Western blotting using anti-GFP antibody. (B) \u003cem\u003eIn vivo\u003c/em\u003e protein degradation rate assay of CP. The leaves of \u003cem\u003eN\u003c/em\u003e. \u003cem\u003ebenthamiana\u003c/em\u003e expressing CP-GFP with TRV-GUS, TRV-ATG3A, TRV-ATG5, TRV-Beclin1, TRV-ATG7A or TRV-ATG8f1 were treated with CHX. The samples were harvested for Western blotting at 0 h and 6 h after treatment. (C) qRT-PCR analysis of the transcript levels of\u003cem\u003e ATG3A, ATG5, Beclin1, ATG7A \u003c/em\u003eand\u003cem\u003e ATG8f1\u003c/em\u003e after TRV-triggered silencing. \u003cem\u003eActin\u003c/em\u003e was used as reference for normalization.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-5886236/v1/05c3590b7d6eaf3fc67b884a.png"},{"id":77232968,"identity":"68cace47-1a88-408f-ba64-510dec50ed3f","added_by":"auto","created_at":"2025-02-26 12:39:11","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":285821,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAPV1 infection or expression of CP induces autophagic activity. \u003c/strong\u003e(A-B)\u003cstrong\u003e \u003c/strong\u003eqRT-PCR analysis of the transcript levels of\u003cem\u003e NbATG3A, NbATG5, ATG7 \u003c/em\u003eand\u003cem\u003e NbATG8\u003c/em\u003ein APV1 infected leaves of \u003cem\u003eN. benthamiana\u003c/em\u003e(A) and \u003cem\u003eAcATG8c-1, AcATG8c-2, AcATG8f AcATG3A1, AcATG3B1, AcATG5A \u003c/em\u003eand\u003cem\u003e AcATG7A \u003c/em\u003ein APV1 infected areca palm (B). (C) APV1 infection induced autophagic activity. Western blotting analysis of NbATG8 in APV1 infected\u003cem\u003e \u003c/em\u003eleaves\u003cem\u003e \u003c/em\u003eof \u003cem\u003eN. benthamiana\u003c/em\u003e using ATG8 specific antibodies. Rubisco was used as loading controls. (D) Representative TEM images of APV1 infected leaves of areca palm. Obvious autophagic structures (red arrows) were observed in the central vacuole of mesophyll cells. Cp: chloroplast, S: starch, V: vacuole. Bars = 5 or 2 μm. (E) Transient expression of CP induced autophagic activity. Western blotting analysis of NbATG8 in \u003cem\u003eN. benthamiana\u003c/em\u003e expressing Flag-CP using ATG8 specific antibodies. Rubisco was used as loading controls.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-5886236/v1/4bb4f1a7daceeea47bba8c5d.png"},{"id":77232976,"identity":"b0c8acb2-2c51-40c8-a3c7-54b527266cc3","added_by":"auto","created_at":"2025-02-26 12:39:11","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":280434,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBoth AcsHSP and AcDNAJB13 interact with AcATG8f1.\u003c/strong\u003e (A) Luciferase Complementation Assay (LCA) examines the interaction between AcATG8f1 and AcDNAJB13 and AcsHSP. Combinations of AcATG8f1-nLuc and AcDnaJB13-cLuc, AcATG8f1-nLuc and EV-cLuc, EV-nLuc and AcDnaJB13-cLuc, or EV-cLuc and EV-nLuc, were infiltrated into quarter part of a leaf of \u003cem\u003eN. benthamiana.\u003c/em\u003e Luciferase activity was determined by the NightShade LB 985 In Vivo Plant Imaging System (Berthold Technologies, Germany) 72 h post-infiltration (hpi). The same assay was applied to analyze the interaction between AcsHSP and AcATG8f1. (B) Bimolecular fluorescence complementation (BiFC) assay demonstrates the interaction of cYFP-AcATG8f1 with both nYFP-AcDnaJB13 and nYFP-AcsHSP. Leaves of \u003cem\u003eN. benthamiana\u003c/em\u003e was collected 72 h post-infiltration (hpi). Bars, 50 μm.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-5886236/v1/42beb5f0b47f534241441ae2.png"},{"id":77232977,"identity":"ce0ae75c-22b4-47a4-8bea-8fe1d1435554","added_by":"auto","created_at":"2025-02-26 12:39:11","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":180345,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSilencing \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eNbsHSP\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eor \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eNbDNAJB13\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e promotes APV1 infection and over expressing \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eAcsHSP\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e inhibits APV1 infection. \u003c/strong\u003eThe expression cassette of full-length APV1 genomic RNA is integrated into a mini-binary vector pCB301 backbone. Upstream 350nt of CP ORF as control element (CE) and GFP CDS sequence were inserted at the junction of the ORF5 and ORF6. \u003cstrong\u003e(A-E)\u003c/strong\u003eSilencing either \u003cem\u003eAcsHSP\u003c/em\u003e or \u003cem\u003eAcDNAJB13 \u003c/em\u003epromotes APV1 infection. TRV-based VIGS vectors with \u003cem\u003eNbsHSP\u003c/em\u003e (118-302bp coding sequence) and\u003cem\u003e NbDNAJB13 \u003c/em\u003e(1-300bp coding sequence) were agroinfiltrated into \u003cem\u003eN. benthamiana\u003c/em\u003e. At 10 days post inoculation (dpi), the constructed GFP-labeled APV1 infectious clone was agroinfiltrated into \u003cem\u003eN. benthamiana\u003c/em\u003e. The APV1 accumulation was indicated by the GFP fluorescence intensity (\u003cstrong\u003eA\u003c/strong\u003e) and density (\u003cstrong\u003eB) \u003c/strong\u003eat 15 dpi\u003cstrong\u003e. \u003c/strong\u003eRT-PCR was used to analyze the expression levelof\u003cem\u003e NbsHSP\u003c/em\u003e and\u003cem\u003e NbDNAJB13\u003c/em\u003e after TRV triggered gene silencing (\u003cstrong\u003eC\u003c/strong\u003e).\u003cstrong\u003e \u003c/strong\u003eWestern-blot was applied to analyze the reporter GFP accumulation using anti-GFP antibody 72 hpi. Mock inoculation and TRV-GUS were used as control \u003cstrong\u003e(D-E)\u003c/strong\u003e. (\u003cstrong\u003eF-H\u003c/strong\u003e) Over expressing\u003cem\u003e AcsHSP\u003c/em\u003einhibits APV1 infection. The transgenic \u003cem\u003eN. benthamiana\u003c/em\u003e over-expressing \u003cem\u003eAcsHSP \u003c/em\u003ewas agroinfiltrated with GFP-labeled APV1 infectious clone. The APV1 accumulation was indicated by the GFP fluorescence intensity (\u003cstrong\u003eF\u003c/strong\u003e) and density (\u003cstrong\u003eG) \u003c/strong\u003eat 20 dpi\u003cstrong\u003e. \u003c/strong\u003eWestern-blot was used to analyze GFP using anti-GFP antibody 72 hpi. WT \u003cem\u003eN. benthamiana \u003c/em\u003ewas used as control (\u003cstrong\u003eH\u003c/strong\u003e).\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-5886236/v1/3e03e67ecce9594ff76cb7c1.png"},{"id":77232990,"identity":"849f7d3f-556d-4db1-8474-05e28fad2ffd","added_by":"auto","created_at":"2025-02-26 12:39:12","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":142281,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA working model summarizing the roles of AcsHSP and AcDNAJB13 in regulation of CP degradation and APV1 infection.\u003c/strong\u003e After APV1 enters plant cells, CP released from disassembled viral particles, along with \u003cem\u003ede novo\u003c/em\u003e synthesized CP, is transported to the nucleus to induce the expression of AcsHSP and AcDNAJB13. The accumulation of viral proteins disrupts ER homeostasis, triggering the unfolded protein response (UPR) and autophagy. AcsHSP and AcDNAJB13 function as cargo receptors to interact with CP at different intracellular locations. The CP is captured by the phagophore through ATG8's interactions with both AcsHSP and AcDNAJB13. The autophagosome containing CP is then delivered to the vacuole for degradation. This then inhibits viral replication by reducing CP accumulation.\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-5886236/v1/5010b9a813836e39a422045e.png"},{"id":77234945,"identity":"7da7a06b-0fbe-4234-a594-3554777cd1bc","added_by":"auto","created_at":"2025-02-26 13:03:11","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4011095,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5886236/v1/0db12589-5d52-402b-acf4-36e668e9dec0.pdf"},{"id":77233478,"identity":"b99b4ecb-a512-4539-8111-3a4f08bbbca1","added_by":"auto","created_at":"2025-02-26 12:47:11","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":5351049,"visible":true,"origin":"","legend":"Dataset1","description":"","filename":"SupplementalMaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-5886236/v1/7497e26e4e123f1fccb75240.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Both small heat shock protein and J-Domain protein direct defense against Areca palm velarivirus 1 (APV1) by degrading coat protein via autophagy pathway","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAutophagy is an evolutionarily conserved cellular mechanism responsible for degrading and recycling damaged or unnecessary intracellular components, essential for maintaining cellular homeostasis \u003csup\u003e1\u003c/sup\u003e. A key feature of autophagy is the formation of double-membrane vesicles known as autophagosomes \u003csup\u003e1, 2, 3\u003c/sup\u003e. These structures originate from the phagophore assembly site (PAS), which primarily derives from the endoplasmic reticulum (ER) \u003csup\u003e4\u003c/sup\u003e. The maturation of autophagosomes from the PAS is regulated by a series of autophagy-related genes (ATGs) \u003csup\u003e5\u003c/sup\u003e, of which, ATG8 is critical for phagophore expansion and closure. ATG8 is conjugated to the membrane lipid phosphatidylethanolamine (PE), a process known as ATG8 lipidation. This lipidation is a hallmark of autophagosome formation and aids in cargo selection \u003csup\u003e6\u003c/sup\u003e. The precursor ATG8 is initially cleaved by the protease ATG4 to expose a C-terminal glycine, enabling subsequent activation by the ATP-dependent enzyme ATG7. This activation facilitates its transfer to ATG3, after which ATG8 is covalently linked to PE by the ATG5-ATG12-ATG16 hexametric complex, which functions as a ligase \u003csup\u003e7, 8\u003c/sup\u003e. ATG8 interacts with several proteins (such as ATG1, Beclin1/ATG6, and ATG7) that are essential for cargo recruitment and autophagosome signaling. Once fully formed, the autophagosome, containing the sequestered damaged organelles and cellular debris, fuses with the vacuole, where its contents are degraded and recycled \u003csup\u003e9, 10, 11\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eEmerging findings highlight the pivotal role of autophagy in plant virus infections \u003csup\u003e1, 12, 13, 14\u003c/sup\u003e. Initially documented by Haxim et al. (2017), autophagy functions as an antiviral mechanism targeting and degrading the βC1 protein of plant geminiviruses through interaction with ATG8 \u003csup\u003e15\u003c/sup\u003e. Hafren et al. (2017) demonstrated that the P4 protein of cauliflower mosaic virus (CaMV) interacts with the autophagy receptor NEIGHBOR OF BRCA1 (NBR1), leading to selective degradation \u003csup\u003e16\u003c/sup\u003e. Turnip mosaic virus (TuMV) encoded NIb protein interacts directly with Beclin1/ATG6, utilizing it as a selective autophagy receptor for NIb degradation \u003csup\u003e17\u003c/sup\u003e. Conversely, some plant viruses suppress or co-opt autophagy to facilitate their infection. For instance, BaMV infection induces the expression of ATGs, utilizing autophagy to selectively engulf viral RNA-containing chloroplasts, aiding viral replication or evading host defenses \u003csup\u003e18\u003c/sup\u003e. The viral F-box protein P0 induces autophagy to degrade membrane-bound ARGONAUTE1 (AGO1) \u003csup\u003e4\u003c/sup\u003e. Barley stripe mosaic virus (BSMV) γb disrupts ATG7-ATG8 interaction, suppressing autophagosome formation and promoting viral infection \u003csup\u003e19\u003c/sup\u003e. VPg of TuMV facilitates the degradation of suppressor of gene silencing 3 (SGS3) by autophagy and ubiquitination \u003csup\u003e20\u003c/sup\u003e. In summary, autophagy exhibits both antiviral and proviral dual roles during plant viral infections, reflecting its complex interplay with viral pathogenesis.\u003c/p\u003e \u003cp\u003eHeat shock proteins (HSPs) are a group of molecular chaperones that respond to stress conditions. They play crucial roles in preventing protein aggregation, and promoting correct protein folding \u003csup\u003e21, 22\u003c/sup\u003e. HSPs vary widely in molecular weight, ranging from approximately 10 to 100 kDa, and are classified into several families including small heat shock proteins (sHSPs), HSP40 (DNAJ or J-Domain proteins), HSP60, HSP70, HSP90, and large heat shock proteins \u003csup\u003e23, 24\u003c/sup\u003e. sHSPs are characterized as 12\u0026ndash;25 kDa polypeptides containing the α-crystallin domain (ACD), which shares homology with the α-crystallins found in the eye lens \u003csup\u003e25, 26, 27\u003c/sup\u003e. In angiosperms, eleven classes of sHSP genes have been identified \u003csup\u003e26, 27\u003c/sup\u003e. J-domain proteins could be classified into four types (I-IV). Type Ⅰ proteins contain a J-domain with a tripeptide HPD motif for interaction with HSP70, in addition, both the zinc-finger motif and the C-terminal domain which are involved in substrate binding, while type Ⅱ retains only the latter and type Ⅲ comprises only the J-domain. Type IV only has a \u0026ldquo;J-like proteins\u0026rdquo;, which lack the HPD motif in the J-domain \u003csup\u003e28\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eWhile HSP family members play roles in promoting or inhibiting viral infections in animal cells, they generally support viral infection in plants \u003csup\u003e29, 30\u003c/sup\u003e. Movement protein of Rice stripe virus (RSV) and Tomato mosaic virus (ToMV) hijack DNAJA proteins (NbMIP1s) to protect itself from autophagic degradation \u003csup\u003e31\u003c/sup\u003e. HSPs interact with viral replicase complexes (VRCs) and other viral macromolecular protein complexes, aiding in viral processes such as replication, translocation, assembly, disassembly, and movement during plant RNA virus infections \u003csup\u003e24, 32, 33, 34\u003c/sup\u003e. In summary, both autophagy and heat shock proteins (HSPs) exhibit dual roles as proviral and antiviral factors in viral infections. However, the coordination between autophagy and HSPs in defending against viral infections is poorly documented, and the underlying mechanisms remain poorly understood.\u003c/p\u003e \u003cp\u003eAreca palm velarivirus 1 (APV1) is a member of the genus \u003cem\u003eVelarivirus\u003c/em\u003e in the family \u003cem\u003eClosteroviridae\u003c/em\u003e, identified in samples from yellowing leaf disease (YLD) affecting \u003cem\u003eAreca catechu\u003c/em\u003e \u003csup\u003e35, 36\u003c/sup\u003e. APV1 is transmitted by the mealybugs, causing yellow leaf symptoms on \u003cem\u003eAreca catechu\u003c/em\u003e \u003csup\u003e37, 38, 39\u003c/sup\u003e. APV1 infection leads to the disassembly of chloroplasts, and the resultant leaf yellowing may serve as a signal for transmission vectors \u003csup\u003e40\u003c/sup\u003e. However, the molecular mechanisms underlying these processes remain largely unknown. To investigate the interaction between APV1 and its plant host, a Yeast Two-Hybrid assay (Y2H) was used to screen for proteins that interact with the coat protein (CP) of APV1, leading to the identification of a sHSP and a DNAJ protein. Further studies revealed that both HSPs function as selective cargo receptors for autophagic degradation of APV1 CP. This represents the first example of both sHSP and J-Domain protein playing an antiviral role via autophagy pathway.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eSmall heat shock protein (AcsHSP) and J-domain protein (AcDNAJB13) interact with APV1 CP\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate potential cellular factors that may interact with APV1, we conducted a yeast two-hybrid (Y2H) screening to identify proteins binding to the APV1 coat protein (APV1-CP). This screening identified ten host candidate proteins that specifically interact with CP (Table S1). Among these, we selected a small heat shock protein, AcsHSP (18.0 kDa), and a J-domain protein, AcDNAJB13 (37.9 kDa), for further investigation. AcsHSP features a 16-amino-acid C-terminal extension (CTE), a typical \u0026alpha;-crystallin domain (ACD) spanning amino acids 50-141, and a 49-amino-acid N-terminal domain (NTD) characterized by a CⅠ NTD motif, \u0026ldquo;IFDPFSLDVWDPF\u0026rdquo; (9-21aa) (Fig. S1A and B). AcsHSP was categorized as a cytosolic class I (CI) protein based on the classification method \u003csup\u003e27\u003c/sup\u003e. AcDNAJB13 contains a J-domain (3-81 aa) featuring the highly conserved tripeptide HPD, which facilitates binding to the ATPase domain of HSP70. Additionally, it has a C-terminal domain (173-336 aa) for substrate binding but lacks a Zn-finger domain (Fig. S1A and C), classifying AcDNAJB13 as a member of the type II J-domain protein family \u003csup\u003e28\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe interaction between AcsHSP-CP and AcDNAJB13-CP was further confirmed through pairwise Y2H assays, which detected strong interactions between AcDNAJB13 and CP, as well as between AcsHSP and CP (Fig. 1A). To validate these interactions\u003cem\u003e\u0026nbsp;in planta\u003c/em\u003e, we conducted bimolecular fluorescence complementation (BiFC) experiments. The host genes AcsHSP and AcDNAJB13 were individually fused to the N-terminal part of YFP, generating nYFP-AcsHSP and nYFP-AcDNAJB13, respectively, while CP was fused to the C-terminal part of YFP to create cYFP-CP. Positive interactions between cYFP-CP and nYFP-AcsHSP, as well as between cYFP-CP and nYFP-AcDNAJB13, were confirmed by the presence of YFP fluorescence, indicating that the two HSPs interact with CP \u003cem\u003ein vivo\u0026nbsp;\u003c/em\u003e(Fig. 1B). Glutathione S-transferase (GST) Pull-down assays confirmed that both GST-AcsHSP and GST-AcDNAJB13 could capture His-tagged CP, demonstrating that both HSPs interact with CP \u003cem\u003ein vitro\u003c/em\u003e (Fig. 1C). These findings were further supported by co-immunoprecipitation (Co-IP) assays, which demonstrated that Myc-AcsHSP and Myc-AcDNAJB13 individually co-precipitated with GFP-CP (Fig. 1D and E). Collectively, these assays confirm that both AcsHSP and AcDNAJB13 interact with CP \u003cem\u003ein vivo\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;in vitro\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eTo identify which domain of AcDNAJB13 is involved in its interaction with CP, we constructed truncated versions of AcDNAJB13 based on the predicted domains and tested them. BiFC assays revealed that the 1\u0026ndash;81 aa and 1\u0026ndash;172 aa fragments interacted with CP, whereas the 82\u0026ndash;345 aa and 173\u0026ndash;345 fragments did not. The interaction pattern of the 1\u0026ndash;81 aa fragment was similar to those of the full-length AcDNAJB13 (Fig. S2), demonstrating that the J-domain of AcDNAJB13 interacts with CP. Furthermore, both AcsHSP and AcDNAJB13 interacted with AcHSP70 (Fig.S3A), deletion of the HPD tripeptide from the J-domain (AcDnaJB13-∆HPD) abolished the interaction with AcHSP70, suggesting that the HPD motif is essential for interaction between the AcDNAJB13 and HSP70 (Fig. S3B). However, AcDnaJB13-∆HPD still interacted with CP in the BiFC assay (Fig. S3C), indicating that the interaction between AcDnaJB13 and CP is independent of the HSP70 chaperone.\u003c/p\u003e\n\u003cp\u003eWe have built an infectious cDNA clone of APV1 which established systemic infection in inoculated \u003cem\u003eN. benthamiana\u003c/em\u003e via agroinoculation, indicating that \u003cem\u003eN. benthamiana\u003c/em\u003e is a compatible host of APV1 (unpublished data). Closest homologs of \u003cem\u003eAcsHSP\u003c/em\u003e and \u003cem\u003eAcDNAJB13\u003c/em\u003e in \u003cem\u003eN. benthamiana\u003c/em\u003e were found by BLAST searches against the SGN database, then cloned and designated hereafter as \u003cem\u003eNbsHSP\u0026nbsp;\u003c/em\u003eand \u003cem\u003eNbDNAJB13\u003c/em\u003e, respectively. BiFC experiments confirmed the interactions of NbsHSP-CP and NbDNAJB13-CP \u003cem\u003ein vivo\u003c/em\u003e (Fig. S4), implying similar roles of NbsHSP and NbDNAJB13 in response to APV1 infection.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCP alters the subcellular localization of AcsHSP\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eViral infections often disturbs the subcellular distribution of host proteins, which affects the plant-virus interaction \u003csup\u003e41\u003c/sup\u003e. Therefore, we examined the intracellular distribution of CP, AcsHSP and AcDNAJB13 to determine whether such changes of subcellular localization occur for AcsHSP and AcDNAJB13 in response to CP expression. First, the CP-GFP fusion protein (N-terminal GFP tag) was transiently expressed in \u003cem\u003eN. benthamiana\u003c/em\u003e. Confocal microscopy showed that CP-GFP was localized in both the cytoplasm and the nucleus in epidermal cells (Fig. 2A). Next, the two HSPs were transiently expressed alone or co-expressed with CP, respectively. Similar to the circumstance of CP, fluorescence of AcsHSP-GFP or AcDNAJB13-GFP alone was detected in both the cytoplasm and the nucleus (Fig. 2B and 2C). Interestingly, when AcsHSP-GFP was transiently expressed alone, multiple punctate fluorescence spots appeared in the cytoplasm. Such punctate spots were disappeared when CP were transiently co-expressed. Apparently, CP altered the intracellular localization of AcsHSP, preventing AcsHSP from forming punctate structures (Fig. 2B). In similar co-expression assays of AcDNAJB13 and CP, the presence of CP seemed to have no effect on the cellular distribution of AcDNAJB13 (Fig. 2C). RFP-tagged AcsHSP or AcDNAJB13 was transiently co-expressed with CP-GFP, overlapped fluorescence was detected in both the cytoplasm and the nucleus, confirmed their interaction and co-localization (Fig. 2B and 2C).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAPV1 infection induces the expression of \u003cem\u003eAcsHSP\u003c/em\u003e and \u003cem\u003eAcDNAJB13\u003c/em\u003e, which conversely impede CP accumulation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo explore the roles of \u003cem\u003eAcsHSP\u003c/em\u003e and\u003cem\u003e\u0026nbsp;AcDNAJB13\u003c/em\u003e in APV1 infection, we used qRT-PCR to analyze changes of their expression patterns in mock-treated and APV1-affected plants. As closteroviruses are known to take a long time (usually two months and longer) to establish systemic infection after inoculation \u003csup\u003e38, 39\u003c/sup\u003e, areca seedlings were inoculated with APV1 via mealybug transmission and sampled at 5 months post-inoculation (mpi), while \u003cem\u003eN. benthamiana\u003c/em\u003e plants were inoculated with a full-length infectious cDNA clone of APV1 via agroinoculation (unpublished data) and sampled at 3 mpi. The results indicated that \u003cem\u003eAcsHSP\u003c/em\u003e and\u003cem\u003e\u0026nbsp;AcDNAJB13\u003c/em\u003e in areca seedlings, as well as their counterparts \u003cem\u003eNbsHSP\u003c/em\u003e and\u003cem\u003e\u0026nbsp;NbDNAJB13\u0026nbsp;\u003c/em\u003ein\u003cem\u003e\u0026nbsp;N. benthamiana\u003c/em\u003e, were all upregulated under systemic infection of APV1 (Fig. 3A-B). Further, Flag-CP transiently expressed in\u003cem\u003e\u0026nbsp;N. benthamiana\u003c/em\u003e enhanced the transcription of \u003cem\u003eNbsHSP\u003c/em\u003e and\u003cem\u003e\u0026nbsp;NbDNAJB13\u0026nbsp;\u003c/em\u003e(Fig. 3C-D)\u003cem\u003e,\u0026nbsp;\u003c/em\u003esuggesting that expression of CP alone is capable to induce the expression of both HSPs.\u003c/p\u003e\n\u003cp\u003eTo assess the effect of induced \u003cem\u003eAcsHSP\u003c/em\u003e and \u003cem\u003eAcDNAJB13\u003c/em\u003e expression on CP, the protein accumulation levels of CP-GFP were examined in \u003cem\u003eN. benthamiana\u003c/em\u003e with or without transiently expressed HSPs. Immunoblotting analysis revealed that the levels of CP-GFP decreased when co-expressed with Myc-AcsHSP (Fig. 3E), as well as when co-expressed with Myc-AcDNAJB13 (Fig. 3F). On the contrary, when a TRV-based virus-induced gene silencing (VIGS) vector was employed to silence \u003cem\u003eNbsHSP\u003c/em\u003e and\u003cem\u003e\u0026nbsp;NbDNAJB13,\u0026nbsp;\u003c/em\u003ethe accumulation level of CP-GFP remarkedly increased, while inoculation of TRV-GUS (served as control) just slightly improved CP accumulation (Fig. 3G and 3H). These results indicate that both AcsHSP and AcDNAJB13 play a key role in CP degradation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDegradation of CP is independent of the UPR pathway\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInfecting virus hijacks translation machinery to produce large amounts of viral proteins, which inevitably perturbs ER homeostasis and often causes unfolded protein response (UPR) \u003csup\u003e42\u003c/sup\u003e. Inspired by that a type-A J-domain proteins NbMIP1s induced by UPR protects RSV protein from autophagic degradation \u003csup\u003e31\u003c/sup\u003e, we investigated whether APV1 infection can induce the UPR. RT-qPCR was applied to analyze the expression of the marker genes for the UPR and ER stress. \u003cem\u003eNbbZIP60, NbbZIP17-1, NbPDI, NbSKP1-1, NbSKP1-4\u003c/em\u003e, and \u003cem\u003eNbbZIP17-2/3\u003c/em\u003e were significantly induced in APV1 infected \u003cem\u003eN. benthamiana, AcbZIP17, AcbZIP60\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;AcbZIP28\u003c/em\u003e were induced in APV1 infected areca palm (Fig S5A), indicating that APV1 infection induces the UPR in plant cells. We further studied whether the degradation of CP is UPR dependent. Previous reports have shown that infiltration of 2mM dithiothreitol (DTT) into \u003cem\u003eN. benthamiana\u003c/em\u003e leaves could efficiently trigger ER stress, activate the UPR and autophagy pathway \u003csup\u003e31\u003c/sup\u003e. However, DTT induced UPR and autophagy did not affect the CP stability (Fig S5B), suggesting that degradation of CP is independent of the UPR pathway.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAPV1 CP is targeted for autophagic degradation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo explore the mechanisms involved in the degradation of CP, we assessed its stability \u003cem\u003ein vivo\u003c/em\u003e by treating \u003cem\u003eN. benthamiana\u0026nbsp;\u003c/em\u003eleaves expressing CP-GFP with cycloheximide (CHX), a known inhibitor of protein synthesis. Following CHX treatment, the synthesis of CP is halted, yet its degradation continues. Immunoblotting revealed a notable decrease in CP-GFP levels within 4 hours of CHX treatment, while free GFP remained stable throughout the observation period (Fig. 4A), indicating that CP is susceptible to degradation within the plant. In eukaryotes, protein degradation primarily occurs through two pathways: the ubiquitin-proteasome and autophagy pathways. To elucidate the degradation pathway of APV1-CP, we employed specific chemical inhibitors targeting both pathways. Leaves expressing APV1-GFP were treated with E64D and 3-MA (inhibitors of autophagy) and MG132 (an inhibitor of the 26S proteasome). Western blot analysis demonstrated that CP-GFP accumulation increased significantly upon treatment with E64D and 3-MA, whereas MG132 treatment did not affect CP levels (Fig. 4B), indicating that CP is primarily degraded via the autophagy pathway.\u003c/p\u003e\n\u003cp\u003eTo investigate whether CP is delivered to autophagosomes, we utilized RFP-tagged AcATG8f1-1 and NbATG8f1 as markers for autophagosomes, examining the localization of CP-GFP. Confocal microscopy showed that CP-GFP colocalized with both RFP-AcATG8f1-1 and RFP-NbATG8f1, forming distinct punctate structures resembling autophagic bodies. These punctate structures were absent in the control group co-expressing CP-GFP with free RFP (Fig. 4C).\u003c/p\u003e\n\u003cp\u003eTo further validate the autophagic degradation of APV1 CP, we silenced key genes involved in cargo recognition and autophagosome signaling using TRV-mediated virus-induced gene silencing (VIGS). Silencing of \u003cem\u003eATG3A, ATG5, Beclin1,\u003c/em\u003e and \u003cem\u003eATG8f1\u003c/em\u003e significantly enhanced the stability of CP-GFP during transient expression (Fig. 5A). We subsequently monitored CP degradation rates\u003cem\u003e\u0026nbsp;in vivo\u003c/em\u003e by treating leaves expressing CP-GFP with CHX and collecting samples at 0 and 6 hours post-treatment (hpt). While \u003cem\u003eTRV-GUS\u003c/em\u003e inoculation (control) led to a moderate increase in CP accumulation, co-expression with TRV constructs targeting \u003cem\u003eNbATG3, NbATG5, NbATG7, NbBeclin1, or NbATG8\u003c/em\u003e resulted in significantly elevated levels of CP-GFP at 6 hpt (Fig. 5B). The silencing efficiency of these genes was confirmed through qRT-PCR (Fig. 5C). Collectively, these findings demonstrate that CP is transported to autophagosomes and undergoes degradation via the canonical autophagy pathway.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAutophagy is induced by APV1 infection or CP expression\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate whether APV1 infection triggers autophagy, we conducted RT-PCR to analyze the expression of autophagic marker genes following APV1 infection. The results showed that all tested marker genes (\u003cem\u003eAcATG8c-1, AcATG8c-2, AcATG8f, AcATG3A1, AcATG3B1, AcATG5A,\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;AcATG7A\u003c/em\u003e) were significantly upregulated in APV1-infected areca palms. In contrast, \u003cem\u003eNbATG3, NbATG5,\u003c/em\u003e and \u003cem\u003eNbATG8c\u003c/em\u003e were induced by APV1 in \u003cem\u003eN. benthamiana\u003c/em\u003e (Fig. 6A-B). Consistent with these findings, Western blot analysis revealed an accumulation of NbATG8-II, a marker of activated autophagy, in samples infected with APV1 (Fig. 6C). To further assess autophagy induction by APV1, we employed transmission electron microscopy (TEM) to monitor autophagic activity. The APV1 infection resulted in a significantly higher number of double-membrane structures characteristic of autophagosomes in the cytoplasm compared to mock-inoculated controls, indicating an enhanced level of autophagic flux in APV1-infected \u003cem\u003eN. benthamiana\u003c/em\u003e (Fig. 6D). We also examined whether autophagy was induced by the CP; transient expression of a Flag-tagged CP in \u003cem\u003eN. benthamiana\u003c/em\u003e led to a strong induction of NbATG8-II, as shown by Western blot analysis, compared to the mock inoculation with an empty vector (Fig. 6E). In summary, these results confirm that autophagy is induced by the CP following APV1 infection. However, it remains unclear whether other APV1-encoded proteins also contribute to this induction.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBoth AcsHSP and AcDNAJB13\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003einteract with AcATG8f1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo determine whether AcsHSP and AcDNAJB13 directly mediate the degradation of CP through the autophagy pathway, we employed the Luciferase Complementation Assay (LCA) to examine the interaction between AcATG8f1 and both AcsHSP and AcDNAJB13. Results demonstrated that both AcsHSP and AcDNAJB13 interacted with AcATG8f1 \u003cem\u003ein planta\u003c/em\u003e, exhibiting strong luciferase activity (Fig. 7A). These interactions were further validated by BiFC assay, which showed that the AcATG8f1-AcsHSP complex dispersed throughout the cytoplasm as abundant irregular granules, while the AcATG8f1-AcDNAJB13 complex predominantly localized to the cytomembrane and nucleus (Fig. 7B). This strongly suggests that AcsHSP and AcDNAJB13 play distinct roles in the autophagy pathway. To identify which domain of AcDNAJB13 interacts with AcATG8f1, we conducted interaction assays using truncated versions of AcDNAJB13. BiFC results indicated that the J-domain (1-81 aa) of AcDNAJB13 interacts with AcATG8f1, with extended peptides (1-171 aa) exhibiting stronger fluorescence. In contrast, the fragment spanning 82-345 aa showed no interaction (Fig. S6), suggesting that the region between 82-172 aa does not directly interact with AcATG8f1 but may regulate or enhance the interaction between the J-domain of AcDNAJB13 and AcATG8f1. These findings indicate that both AcsHSP and AcDNAJB13 may server as cargo receptors for CP degradation in the autophagy pathway.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe interactions between CP and both AcsHSP and AcDNAJB13 are required for CP degradation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo further determine whether the interactions between CP and both AcsHSP and AcDNAJB13 are necessary for CP degradation, BiFC assay was first used to identify which truncated fragment of CP interacts with AcsHSP and AcDNAJB13. BiFC revealed that the 1-110 aa fragment of CP interacted with both AcsHSP and AcDNAJB13, whereas the 111-205aa and 205-298aa fragments did not (Fig. S7A and S7B). Then, the truncated Frag-CP (111-298aa) was transiently co-expressed with myc-AcsHSP and myc-AcDNAJB13 in\u0026nbsp;\u003cem\u003eN. benthamiana\u003c/em\u003e, respectively. Seedlings were treated with CHX at 48 h post agroinfiltration. Samples were collected at 12 hours post-treatment (hpt). Western-blot indicated that the stability of truncated CP was not affected by expression of \u003cem\u003emyc-AcsHSP\u003c/em\u003e (Fig. S7C) or \u003cem\u003emyc-AcDNAJB13\u0026nbsp;\u003c/em\u003e(Fig. S7D)\u003cem\u003e,\u003c/em\u003e indicating that the interactions between CP and both AcsHSP and AcDNAJB13 are required for CP degradation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSilencing \u003cem\u003eNbsHSP\u0026nbsp;\u003c/em\u003eor \u003cem\u003eNbDNAJB13\u003c/em\u003e promotes APV1 infection and over expressing \u003cem\u003eAcsHSP\u003c/em\u003e inhibits APV1 infection.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo determine whether the expression levels of \u003cem\u003eNbsHSP\u003c/em\u003e or \u003cem\u003eNbDNAJB13\u003c/em\u003e influence APV1 infection, TRV-induced VIGS was used to silence \u003cem\u003eNbsHSP\u003c/em\u003e or \u003cem\u003eNbDNAJB13,\u003c/em\u003e then after, a GFP-labeled infectious clone for APV1 was constructed and agroinfiltrated into \u003cem\u003eN. benthamiana\u003c/em\u003e to monitor the APV1 infection. TRV-triggered Silencing either \u003cem\u003eNbsHSP\u003c/em\u003e or \u003cem\u003eNbDNAJB13\u003c/em\u003e significantly reduced the expression levels of \u003cem\u003eNbsHSP\u003c/em\u003e or \u003cem\u003eNbDNAJB13\u003c/em\u003e (Fig. 8C) and promoted APV1 infection indicating by the GFP fluorescence intensity (Fig. 8A)\u0026nbsp;and density (Fig. 8B), which was further verified by the Western blotting using antibody against GFP (Fig. 8D and 8E). In contrast, overexpressing \u003cem\u003eACsHSP\u003c/em\u003e in \u003cem\u003eN. benthamiana\u003c/em\u003e substantially inhibited APV1 infection (Fig. 8F-H). These results strongly suggest that both sHSP and DNAJB13 play a role in the antiviral defense against APV1 infection through CP degradation via the autophagy pathway.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eHeat shock proteins (HSPs) exhibit both antiviral and proviral functions in viral infections of humans and animals \u003csup\u003e23, 43\u003c/sup\u003e. While there is limited documentation of their antiviral functions in plant, and the underlying mechanisms remain largely unexplored \u003csup\u003e29, 30\u003c/sup\u003e. This study presents novel findings on the roles of both sHSP and J-Domain proteins in mediating defense against plant virus through an autophagic degradation pathway. Based on protein structure predictions, 18.0 kD AcsHSP is categorized as a CI class sHSP, confirmed to localize in both the cytoplasm and the nucleus. CI sHSPs, along with HSP101, have been shown to protect eukaryotic translation initiation factors during heat stress (McLoughlin et al., 2016). However, there has yet to be any report of CI sHSPs participating in viral infections. Notably, the 23.6 kD sHSP from \u003cem\u003eTriticum aestivum\u003c/em\u003e has been found to interact with the coat protein of the wheat yellow mosaic virus \u003csup\u003e44\u003c/sup\u003e, while the 22.8 kD sHSP from \u003cem\u003eCitrullus lanatus\u003c/em\u003e interacts with the helicase of cucumber green mottle mosaic virus (CGMMV). Overexpression of this sHSP was associated with reduced viral RNA accumulation and slowed disease progression, while silencing the sHSP did not significantly affect CGMMV infection \u003csup\u003e41\u003c/sup\u003e, and the underlying mechanism remain unexplored. AcsHSP is the first example of sHSP playing an antiviral role via autophagy pathway.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDNAJ/Hsp40 proteins are well-established as having both antiviral and proviral functions during infections caused by human viruses (Multhoff, 2006). For instance, the type I J-domain protein DNAJA3 has been shown to interact with the VP1 protein of the foot-and-mouth disease virus (FMDV), leading to its degradation through the autophagy pathway \u003csup\u003e45\u003c/sup\u003e. Conversely, the type II J-domain protein Hdj1 interacts with the core protein of hepatitis B virus, inhibiting viral replication by destabilizing viral proteins \u003csup\u003e46\u003c/sup\u003e. Another type II J-domain protein, DnaJB1, facilitates the replication of influenza A virus by aiding the nuclear import of viral ribonucleoproteins \u003csup\u003e47\u003c/sup\u003e. Type III J-domain protein DNAJC14 has been found to inhibit yellow fever virus and hepatitis C virus during the viral RNA replication phase \u003csup\u003e48\u003c/sup\u003e. In plant, type I J-domain proteins NbMIP1s interact with both the viral movement protein and the resistance protein Tm-2\u003csup\u003e2\u003c/sup\u003e. Their silencing compromises the stability of both proteins, exhibiting a dual functionality in both the resistance and the facilitation to virus infection \u003csup\u003e49\u003c/sup\u003e. The NbMIP1s interacts with RSV NSvc4, preventing its autophagic degradation and thus promoting viral infection \u003csup\u003e31\u003c/sup\u003e. A type III J-domain protein GmHSP40 identified in soybean plays a positive role in viral defense; its overexpression induces hypersensitive response-like cell death, while its silencing increases susceptibility to Soybean mosaic virus \u003csup\u003e50\u003c/sup\u003e. These findings highlight the complex roles of J-domain proteins in the ongoing conflict between viruses and plant hosts. This study focuses on the type II J-domain protein AcDNAJB13, which interacts with both viral CP and ATG8f, indicating that AcDNAJB13 is a selective cargo receptor for the degradation of CP through the autophagy pathway.\u003c/p\u003e\n\u003cp\u003eAcsHSP and AcDNAJB13 do not possess any shared functional domains or sequence homology, prompting an intriguing inquiry into their similar roles in defense against APV1. Specifically, both HSPs have been shown to interact with CP (Fig. 1 and Fig. S4). Moreover, their respective gene expressions are upregulated following APV1 infection as well as the expression of CP (Fig. 3A-B). Additionally, both AcsHSP and AcDNAJB13 interact with ATG8f (Fig. 7) and are crucial for the autophagic degradation of CP (Fig. 3C-D). The closest homologs found in \u003cem\u003eN. benthamiana\u003c/em\u003e, namely NbsHSP and NbDNAJB13, demonstrate a similar defensive role against APV1, suggesting that these two types of HSPs are conserved across the plant kingdom. Nevertheless, different functions between AcsHSP and AcDNAJB13 could be reflected by their cellular localization. On the one hand, the subcellular localization of AcsHSP was altered by CP appearance, while AcDNAJB13 was not (Fig. 2). On the other hand, BiFC revealed AcATG8f1-AcsHSP complex dispersed throughout the cytoplasm as abundant irregular granules, while the AcATG8f1-AcDNAJB13 complex predominantly localized to the cytomembrane and nucleus (Fig. 7B). These results indicate the distinct roles of AcsHSP and AcDNAJB13 playing in the plant cell, it requires further investigation.\u003c/p\u003e\n\u003cp\u003eAutophagy can act as an antiviral mechanism by directly targeting viral proteins for degradation through ATG8, as seen with \u0026beta;C1 of geminiviruses \u003csup\u003e51\u003c/sup\u003e, C1 proteins from tomato leaf curl Yunnan virus (TLCYnV), and various other geminiviruses \u003csup\u003e12, 52\u003c/sup\u003e. In another autophagy pathway, cargo receptor is required to assist ATG8 in capturing viral proteins for degradation. For instance, NBR1 serves as a cargo receptor that targets the capsid protein P4 of Cauliflower mosaic virus (CaMV) for autophagic degradation\u0026nbsp;\u003csup\u003e16\u003c/sup\u003e. NbP3IP directs the autophagic degradation of RSV p3 by interacting with both p3 and NbATG8f\u0026nbsp;\u003csup\u003e53\u003c/sup\u003e. Virus-induced small peptide 1 (VISP1) acts as a cargo receptor exhibiting antiviral activity by mediating the autophagic degradation of various viral proteins, including CMV 2b, the P14 protein of pothos latent virus (PoLV), the C2 protein of beet severe curly top virus (BSCTV), and the AC2 protein of cabbage leaf curl virus (CaLCuV)\u0026nbsp;\u003csup\u003e54, 55\u003c/sup\u003e. This work represents the first example that both sHSP and J-Domain protein function as selective autophagosomal cargo receptors for the autophagic degradation of viral protein, restricting virus infection.\u003c/p\u003e\n\u003cp\u003eIn summary, we propose a model illustrating the roles of AcsHSP and AcDNAJB13 in the APV1 infection cycle (Fig. 9). APV1 infection activates autophagy to target CP for degradation. APV1 also triggers the ER stress and the UPR. However, the degradation of CP is independent on UPR. The CP is captured intracellularly by both AcsHSP and AcDNAJB13, which is further recruited to the phagophore through ATG8\u0026apos;s interactions with AcsHSP and AcDNAJB13. AcsHSP and AcDNAJB13 function as cargo receptors for autophagy. This work highlights the novel roles of sHSP and J-domain protein, offering new insights into antiviral function of HSP families.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003ePlant Materials and Virus Inoculation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAreca palm seedlings were inoculated with mealybugs (\u003cem\u003eFerrisia virgata\u003c/em\u003e or \u003cem\u003ePseudococcus cryptus\u003c/em\u003e) carrying APV1, following previously established protocols \u003csup\u003e38, 39\u003c/sup\u003e. \u003cem\u003eNicotiana benthamiana\u003c/em\u003e was inoculated using an infectious cDNA clone of APV1 through agroinoculation. Transgenic lines of \u003cem\u003eN. benthamiana\u003c/em\u003e expressing the coat protein (CP) of APV1 are maintained in our laboratory. All plants were cultivated in a growth chamber under controlled conditions of 25\u0026deg;C with a 16-hour light period and 18\u0026deg;C with an 8-hour dark period, maintaining a relative humidity of 75%.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSubcellular Localization\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAcsHSP, AcDNAJB13,\u0026nbsp;\u003c/em\u003eand \u003cem\u003eCP\u003c/em\u003e were amplified via RT-PCR and cloned into the pCambia1302-GFP vector, resulting in constructs pCambia1302-GFP-AcsHSP, pCambia1302-GFP-AcDNAJB13, and pCambia1302-GFP-CP. Additionally, the coding sequences of \u003cem\u003eAcsHSP\u003c/em\u003e and \u003cem\u003eAcDNAJB13\u003c/em\u003e were fused with the RFP protein to create constructs 35S::RFP-AcsHSP and 35S::RFP-AcDNAJB13. The vectors were infiltrated into the leaves of \u003cem\u003eN. benthamiana\u003c/em\u003e, and fluorescence was observed using a Zeiss LSM 780 confocal microscope at 72 hours post-inoculation (hpi). All primers used in this study are listed in Table S2. Gene amplification was performed using PrimeSTAR\u0026reg; GXL DNA Polymerase (TaKaRa, Dalian), and all vectors were constructed using the ClonExpress\u0026reg; Ultra One Step Cloning Kit (Vazyme, Nanjing, China, Cat #C115).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eYeast Two-Hybrid (Y2H) Screening\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo identify proteins that interact with APV1 CP, a Y2H cDNA library from areca palm was constructed by OE Biotech (Shanghai, China). The bait vector pGBKT7-CP was created, and Y2H screening was conducted using the Yeastmaker\u0026trade; Yeast Transformation System 2 (Clontech, USA). To confirm the interaction candidates in yeast, the prey vectors pGADT7-AcsHSP and pGADT7-AcDNAJB13 were co-transformed with the bait vector pGBKT7-CP into the yeast strain Y187. The transformed yeast was plated on minimal media with triple dropouts (TDO, SD/-Trp/-Leu/-His) and quadruple dropouts (QDO, SD/-Trp/-Leu/-His/-Ade) supplemented with AbA and X-\u0026alpha;-Gal, respectively. pGBKT7-53 and pGADT7-T served as positive controls, while pGBKT7-Lam and pGADT7-T served as negative controls. Three independent experiments were conducted to validate the results.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBimolecular Fluorescence Complementation (BiFC) and Complementation Luciferase Assay (CLA) in Planta\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe BiFC assay was conducted as previously described \u003csup\u003e31\u003c/sup\u003e. Vectors pFGC-YN173-AcsHSP, pFGC-YN173-AcDNAJB13, and pFGC-CN155-CP were created. The YN-CN combinations of vectors were agroinfiltrated into \u003cem\u003eN. benthamiana\u003c/em\u003e leaves, and epidermal cells from 1-cm infiltrated leaf explants were collected at 72 hpi for observation using a Zeiss LSM 780 confocal microscope. CLA assays were performed as previously outlined \u003csup\u003e56\u003c/sup\u003e, with constructs AcDNAJB13-cLUC, AcsHSP-cLUC, and AcATG8f1-nLUC. The nLUC/cLUC vector combinations were co-infiltrated into \u003cem\u003eN. benthamiana\u003c/em\u003e leaves through agroinoculation. Leaves were harvested at 48 hpi, treated with 1 mM luciferin, and imaged using the NightShade LB 985 In Vivo Plant Imaging System (Berthold Technologies, Germany) two minutes after luciferin treatment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eIn Vitro\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;GST Pull-Down Assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe GST pull-down assay was performed according to previously established methods \u003csup\u003e41\u003c/sup\u003e. The coding sequences of AcsHSP and AcDNAJB13 were cloned into the pGEX-6p vector, while the open reading frame (ORF) of CP was cloned into the pET28a vector. GST-AcsHSP, GST-AcDNAJB13, and His-CP were expressed in E. coli BL21 (DE3) and purified using a GST-tag and His-tag Protein Purification Kit (Beyotime, Shanghai, China, Cat #P2226). The pull-down assay was conducted using the BersinBio\u0026trade; GST-pulldown Kit (Cat #Bes3012) following the manufacturer\u0026apos;s protocol. For each assay, 2 \u0026mu;g of His-CP protein was incubated with equal amounts of GST-AcDNAJB13, GST-AcsHSP, and GST on BersinBio GSH magnetic beads at 4\u0026deg;C for 12 hours. Proteins bound to the beads were collected, separated by SDS-PAGE, and analyzed by Western blot using anti-GST (TransGen Biotech, Beijing, China, Cat #HT601) and anti-His antibodies (TransGen Biotech, Beijing, China, Cat #HT501).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIn Vivo Co-Immunoprecipitation (Co-IP) Assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor Co-IP assays, plasmids pCambia1302-GFP-CP in combination with pCambia1302-Myc-AcHsp or pCambia1302-Myc-AcDNAJB13 were agroinfiltrated into \u003cem\u003eN. benthamiana\u003c/em\u003e. The infiltrated leaves were harvested at 72 hpi, and total proteins were extracted from frozen, ground tissue using ice-cold extraction buffer (50 mM HEPES, pH 7.5, 150 mM KCl, 1 mM EDTA, and 0.5% Triton X-100), supplemented with 1 mM dithiothreitol and a 1\u0026times; protease inhibitor cocktail (Roche) \u003csup\u003e53\u003c/sup\u003e. After centrifugation at 12,000 g for 10 minutes at 4\u0026deg;C, the supernatant was collected and incubated with Anti-GFP Magnetic Beads (Yamei, Shanghai, China, Cat #YJ010) at room temperature for one hour with gentle agitation. The precipitates were washed three times with IP buffer and analyzed by Western blotting using anti-Myc antibodies.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eVirus-Induced Gene Silencing (VIGS)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTobacco rattle virus (TRV)-based vectors pTRV2 containing partial gene fragments of \u003cem\u003eNbsHSP, NbsDNAJB13, NbATG3A, NbATG5, NbBeclin1, NbATG7A, NbATG8f1\u003c/em\u003e, or \u003cem\u003eGUS\u0026nbsp;\u003c/em\u003ewere constructed and introduced into\u003cem\u003e\u0026nbsp;Agrobacterium tumefaciens\u003c/em\u003e strain GV3101. Agrobacterium cultures carrying TRV1 or TRV2-derived vectors were mixed in infiltration buffer (10 mM MgCl₂, 10 mM MES, and 200 mM acetosyringone) and infiltrated into \u003cem\u003eN. benthamiana.\u003c/em\u003e The infiltrated plants were used for further analysis at 10 days post-inoculation (dpi).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern Blotting\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal proteins from plant leaf samples were extracted using lysis buffer (50 mM Tris-HCl, pH 6.8, 4.5% SDS, 7.5% \u0026beta;-mercaptoethanol, 9 M urea). After centrifugation at 10,000 \u0026times; g for 15 minutes at 4\u0026deg;C, the supernatant was collected for Western blot assays. Proteins were detected using monoclonal antibodies against GFP (1:5000; HT801; TransGen, Beijing, China), His (1:5000; HT501; TransGen), GST (1:5000; HT601; TransGen), and Flag (1:5000; R24091; ZEN-BIOSCIENCE, Chengdu, China), as well as anti-Myc (1:5000; 341173; ZEN-BIOSCIENCE). Monoclonal antibodies served as primary antibodies, while horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (1:5000; HS201; TransGen) or HRP-conjugated goat anti-rabbit IgG (1:5000; AS014; ABclonal, Wuhan, China) were used as secondary antibodies. Blots were visualized with a chemiluminescence film (Thermo Fisher SuperSignal West Pico PLUS Chemiluminescent Substrate, catalog no. 34577).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eChemical Treatment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eChemical treatments were performed as previously described \u003csup\u003e31\u003c/sup\u003e. Inhibitors of protein degradation pathways, including MG132 (cat: HY-13259, MCE, Monmouth Junction, USA), 3-MA (cat: HY-19312, MCE), and E64d (cat: HY-100229, MCE), were dissolved in DMSO and diluted in ddH₂O to final concentrations of 100 \u0026mu;M, 5 mM, and 100 \u0026mu;M, respectively. An equal volume of DMSO diluted in ddH₂O served as the control. CHX (cat: HY-12320, MCE) and DTT were directly dissolved in ddH₂O to final concentrations of 100 \u0026mu;M and 2 mM, respectively. After infiltration, leaves were kept moist for 12 hours and then harvested for Western blotting.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConfocal Microscopy\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ezonfocal imaging was conducted as described previously \u003csup\u003e57\u003c/sup\u003e. The eGFP fluorophore was excited at 488 nm with emission detected at 490-540 nm; the YFP fluorophore was excited at 514 nm with emission detected at 520-560 nm; and the RFP fluorophore was excited at 561 nm with emission detected at 560-620 nm. Images were captured using a laser scanning confocal microscope (LSM 780, Carl Zeiss) and processed with LSM software (Zen 2012, Carl Zeiss).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTransmission Electron Microscopy (TEM)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTEM was performed as previously described \u003csup\u003e40\u003c/sup\u003e. Briefly, betel palm leaves were cut into 2 mm \u0026times; 2 mm pieces and fixed overnight at 4\u0026deg;C in a fixation buffer containing 2.5% glutaraldehyde and 0.05 M phosphate (pH 7.2). Samples were washed three times with fixation buffer and post-fixed in 2% OsO₄ at 4\u0026deg;C for two hours. Following dehydration through a graded ethanol series (50%, 70%, 80%, 90%, 95%, and 100%), tissue samples were embedded in Spurr\u0026rsquo;s resin. Ultrathin sections (70\u0026ndash;90 nm) were prepared using a Leica EM UC7 ultramicrotome and stained sequentially with uranyl acetate for 20 minutes and Reynolds\u0026rsquo; lead citrate for 5 minutes. Sections were observed using a Hitachi H-7650 or a JEM-1230 transmission electron microscope operating at 80 kV.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQuantitative Reverse Transcription PCR (qRT-PCR)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was isolated using the Tiangen plant RNA isolation kit (DP441, Tiangen Biotech, Beijing, China). Reverse transcription of total RNA was performed using the Thermo Scientific RevertAid First Strand cDNA Synthesis Kit (K1622, Thermo Fisher Scientific, USA). qRT-PCR was conducted using TransStart Tip Green qPCR SuperMix (AQ141, TransGen Biotech, China). AcActin and NbActin were used as internal controls for areca and \u003cem\u003eN. benthamiana\u003c/em\u003e, respectively. All experiments were performed with at least three biological replicates, and relative gene expression levels were calculated using the 2-\u0026Delta;\u0026Delta;Ct method. Primer sets used are listed in Table S2.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experiments were conducted at least three times unless otherwise specified. Statistical differences between samples were analyzed using a two-tailed, unpaired Student\u0026rsquo;s t-test with equal variance (computed in Microsoft Excel). Differences were considered statistically significant at P \u0026lt; 0.05, indicated in figure legends as *, P \u0026lt; 0.05 or **, P \u0026lt; 0.01.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAccession numbers\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGene sequences from this study can be found in GenBank under the following accession numbers: \u003cem\u003eAcsHSP\u003c/em\u003e, PQ623096; \u003cem\u003eAcDNAJB13\u003c/em\u003e, PQ623095; \u003cem\u003eAcATG8f1\u003c/em\u003e; \u003cem\u003eNbsHSP\u003c/em\u003e, PQ623098; \u003cem\u003eNbDNAJB13\u003c/em\u003e, PQ623097; \u003cem\u003eNbATG3A,\u003c/em\u003e KX369396; \u003cem\u003eNbATG5,\u003c/em\u003e KX369397; \u003cem\u003eNbBeclin1\u003c/em\u003e, AY701316; \u003cem\u003eNbATG7A\u003c/em\u003e, KX369398; \u003cem\u003eNbATG8f1\u003c/em\u003e; MG733106.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by grants from National Natural Science Foundation of China (Grants Nr. 32460408), Hainan Key Research and Development Plan Foundation (ZDYF2024XDNY208) and the earmarked fund for Agriculture Research System in Hainan Province (Grant No. HNARS-1-G4-1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eX.H. and R.Z. designed all experiments. X.C., J.L., Z.X., and H.W. performed the protein interaction experiments, virus inoculation, and blotting experiments. X.C. and L.J. performed the experiments for immunofluorescence staining and electron microscopy. H.W. and J.Z. analyzed the data. X.H. and R.Z. organized the project and wrote the manuscript. All authors read and approved the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data that support the findings of this study are available from the corresponding author upon request. Source data are provided with this paper.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eYang M, Liu Y. 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Geminiviral C2 proteins inhibit active autophagy to facilitate virus infection by impairing the interaction of ATG7 and ATG8. \u003cem\u003eJournal of integrative plant biology\u003c/em\u003e \u003cstrong\u003e65\u003c/strong\u003e, 1328-1343 (2023).\u003c/li\u003e\n\u003cli\u003eLi F\u003cem\u003e, et al.\u003c/em\u003e A calmodulin-like protein suppresses RNA silencing and promotes geminivirus infection by degrading SGS3 via the autophagy pathway in Nicotiana benthamiana. \u003cem\u003ePLoS pathogens\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, e1006213 (2017).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Areca palm velarivirus 1, autophagy, small heat shock protein, J-domain protein, cargo receptor","lastPublishedDoi":"10.21203/rs.3.rs-5886236/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5886236/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBoth autophagy and heat shock proteins (HSPs) play dual roles in promoting or inhibiting viral infections. However, the coordination between autophagy and HSPs in the defense against viral infections remains underexplored, and the underlying mechanisms are still poorly understood. This study first revealed an interaction between a cytosolic small heat shock protein (AcsHSP) and a type II J-domain protein (AcDNAJB13) of areca palm with the coat protein (CP) of Areca Palm Velarivirus 1 (APV1) and the interaction is independent of the HSP70 chaperones. The closest homologs in \u003cem\u003eNicotiana benthamiana\u003c/em\u003e (NbsHSP and NbDNAJB13) also interacted with CP. Both AcsHSP and AcDNAJB13 were localized in the cytoplasm and nucleus, and co-expression with CP altered AcsHSP intracellular localization. APV1 infection or transient CP expression induced the expression of \u003cem\u003eAcsHSP\u003c/em\u003e and \u003cem\u003eAcDNAJB13\u003c/em\u003e, which, in turn, inhibited CP accumulation. Virus-induced gene silencing (VIGS) of \u003cem\u003eNbsHSP\u003c/em\u003e and \u003cem\u003eNbDNAJB13\u003c/em\u003e significantly increased the accumulation of transiently expressed CP-GFP. CP degradation occurred via an autophagic pathway. Both AcsHSP and AcDNAJB13 interacting with AcATG8f1, and these interactions were required for CP degradation. Furthermore, silencing endogenous \u003cem\u003eNbsHSP\u003c/em\u003e and \u003cem\u003eNbDNAJB13\u003c/em\u003e enhanced APV1 replication, while overexpression of \u003cem\u003eAcsHSP\u003c/em\u003e reduced APV1 accumulation. Our findings demonstrate that AcsHSP and AcDNAJB13 function as selective cargo receptors for CP degradation via autophagy pathway, thereby limiting APV1 infection and offering new insights into the roles of heat shock protein families.\u003c/p\u003e","manuscriptTitle":"Both small heat shock protein and J-Domain protein direct defense against Areca palm velarivirus 1 (APV1) by degrading coat protein via autophagy pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-02-26 12:39:05","doi":"10.21203/rs.3.rs-5886236/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"147ace5d-c725-4ecf-a292-af04bfc164c0","owner":[],"postedDate":"February 26th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":44643799,"name":"Biological sciences/Microbiology/Virology/Viral pathogenesis"},{"id":44643800,"name":"Biological sciences/Plant sciences/Plant immunity/Effectors in plant pathology"}],"tags":[],"updatedAt":"2026-04-17T13:43:13+00:00","versionOfRecord":[],"versionCreatedAt":"2025-02-26 12:39:05","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5886236","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5886236","identity":"rs-5886236","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}