HC-Pro protein of Potato virus Y can interact with three Arabidopsis 20S proteasome subunits in planta.
ABSTRACT: The multifunctional protein helper component proteinase (HC-Pro) is thought to interfere with the activity of the 20S proteasome; however, no sites of interaction have been identified for either protein. Here, we first show that the Potato virus Y (PVY) HC-Pro protein can interact with three Arabidopsis 20S proteasome subunits (PAA, PBB, and PBE), using a yeast two-hybrid system and the bimolecular fluorescence complement assay. In addition, yeast two-hybrid analysis of the interaction between several mutant subunits of the 20S proteasome and PVY HC-Pro confirmed that residues 81 to 140 of PAA, 1 to 80 of PBB, and 160 to 274 of PBE are necessary for binding PAA, PBB, and PBE to PVY HC-Pro, respectively. Deletion mutant analysis of PVY HC-Pro showed that the N terminus (residues 1 to 97) is necessary for its interaction with three Arabidopsis 20S proteasome subunits. The ability of HC-Pro to interact and interfere with the activity of the 20S proteasome may help explain the molecular basis of its multifunctional character.
Project description:The ubiquitin/26S proteasome system plays an essential role not only in maintaining protein turnover, but also in regulating many other plant responses, including plant-pathogen interactions. Previous studies highlighted different roles of the 20S proteasome in plant defense during virus infection, either indirectly through viral suppressor-mediated degradation of Argonaute proteins, affecting the RNA interference pathway, or directly through modulation of the proteolytic and RNase activity of the 20S proteasome, a component of the 20S proteasome, by viral proteins, affecting the levels of viral proteins and RNAs. Here we show that MG132, a cell permeable proteasomal inhibitor, caused an increase in papaya ringspot virus (PRSV) accumulation in its natural host papaya (Carica papaya). We also show that the PRSV HcPro interacts with the papaya homologue of the Arabidopsis PAA (?1 subunit of the 20S proteasome), but not with the papaya homologue of Arabidopsis PAE (?5 subunit of the 20S proteasome), associated with the RNase activity, although the two 20S proteasome subunits interacted with each other. Mutated forms of PRSV HcPro showed that the conserved KITC54 motif in the N-terminal domain of HcPro was necessary for its binding to PAA. Co-agroinfiltration assays demonstrated that HcPro expression mimicked the action of MG132, and facilitated the accumulation of bothtotal ubiquitinated proteins and viral/non-viral exogenous RNA in Nicotiana benthamiana leaves. These effects were not observed by using an HcPro mutant (KITS54), which impaired the HcPro - PAA interaction. Thus, the PRSV HcPro interacts with a proteasomal subunit, inhibiting the action of the 20S proteasome, suggesting that HcPro might be crucial for modulating its catalytic activities in support of virus accumulation.
Project description:The photosynthetic rate of virus-infected plants is always reduced. However, the molecular mechanism underlying this phenomenon remains unclear. The helper component-proteinase (HC-Pro) of Potato virus Y (PVY) was found in the chloroplasts of PVY-infected tobacco, indicating some new function of HC-Pro in the chloroplasts. We generated HC-Pro transgenic plants with a transit peptide to target the protein to chloroplast. The HC-Pro transgenic tobacco showed a decreased photosynthetic rate by 25% at the light intensity of 600??mol m(-2) s(-1). Using a yeast two-hybrid screening assay to search for chloroplast proteins interacting with HC-Pro, we identified that PVY HC-Pro can interact with the chloroplast ATP synthase NtCF1?-subunit. This interaction was confirmed by GST pull-down and co-immunoprecipitation assays. HC-Pro didn't interfere with the activity of assembled ATP synthase in vitro. The HC-Pro/NtCF1?-subunit interaction might affect the assembly of ATP synthase complex. Quantitative western blot and immunogold labeling of the ATP synthase indicated that the amount of ATP synthase complex was decreased in both the HC-Pro transgenic and the PVY-infected tobacco. These results demonstrate that HC-Pro plays an important role in reducing the photosynthetic rate of PVY-infected plants, which is a completely new role of HC-Pro besides its multiple known functions.
Project description:Potato virus Y (PVY) is an important plant virus and causes great losses every year. Viral infection often leads to abnormal chloroplasts. The first step of chloroplast division is the formation of FtsZ ring (Z-ring), and the placement of Z-ring is coordinated by the Min system in both bacteria and plants. In our lab, the helper-component proteinase (HC-Pro) of PVY was previously found to interact with the chloroplast division protein NtMinD through a yeast two-hybrid screening assay and a bimolecular fluorescence complementation (BiFC) assay in vivo. Here, we further investigated the biological significance of the NtMinD/HC-Pro interaction. We purified the NtMinD and HC-Pro proteins using a prokaryotic protein purification system and tested the effect of HC-Pro on the ATPase activity of NtMinD in vitro. We found that the ATPase activity of NtMinD was reduced in the presence of HC-Pro. In addition, another important chloroplast division related protein, NtMinE, was cloned from the cDNA of Nicotiana tabacum. And the NtMinD/NtMinE interaction site was mapped to the C-terminus of NtMinD, which overlaps the NtMinD/HC-Pro interaction site. Yeast three-hybrid assay demonstrated that HC-Pro competes with NtMinE for binding to NtMinD. HC-Pro was previously reported to accumulate in the chloroplasts of PVY-infected tobacco and we confirmed this result in our present work. The NtMinD/NtMinE interaction is very important in the regulation of chloroplast division. To demonstrate the influence of HC-Pro on chloroplast division, we generated HC-Pro transgenic tobacco with a transit peptide to retarget HC-Pro to the chloroplasts. The HC-Pro transgenic plants showed enlarged chloroplasts. Our present study demonstrated that the interaction between HC-Pro and NtMinD interfered with the function of NtMinD in chloroplast division, which results in enlarged chloroplasts in HC-Pro transgenic tobacco. The HC-Pro/NtMinD interaction may cause the formation of abnormal chloroplasts in PVY-infected plants.
Project description:The three-dimensional fold of Plasmodium falciparum (Pf) 20S proteasome is similar to yeast Saccharomyces cerevisiae 20S proteasome. The twenty eight subunits complex corresponding to two copies of seven distinct ? and seven distinct ? subunits shares >35% sequence identity with equivalent subunits of the yeast 20S proteasome. Bortezomib (Velcade®) - a known inhibitor of the three catalytic subunits; ?1, ?2, ?5 of the yeast 20S proteasome can bind in the equivalent subunits of the Pf 20S proteasome and is in agreement with experimental results. The model defines the binding mode of the bortezomib inhibitor within the catalytic subunits of the Pf 20S proteasome and provides the structural basis for the design of Pf 20S proteasome-specific inhibitors. The substitutions associated within the catalytic subunits of Pf 20S proteasome relative to yeast 20S proteasome; Thr21-Ser, Thr22-Ser, Thr31-Ser, Thr35-Asn, Ala49-Ser (in ?1 subunit), Ser20-Ala, Gln22-Glu (?2) and Thr21-Ser, Ala22-Met, Gln53-Leu (?5) may influence the relative caspase-like, tryptic-like and chymotryptic-like activities of the Pf 20S proteasome. The plasmodia-specific 'large' insert comprising fifty four amino acid residues (in ?1 subunit) of the Pf 20S proteasome is distant from the catalytic sites.
Project description:Two tobacco vein necrosis (TVN) determinants, the residues K(400) and E(419) , have been identified previously in the helper component-protease (HC-Pro) protein sequence of Potato virus Y (PVY). However, since their description, non-necrotic PVY isolates with both K(400) and E(419) necrotic determinants have been reported in the literature. This suggests the presence in the viral genome of other, as yet uncharacterized, TVN determinant(s). The identification of PVY(N) pathogenicity determinants was approached through the replacement of genomic regions of the necrotic PVY(N) -605 infectious clone by corresponding sequences from the non-necrotic PVY(O) -139 isolate. Series of PVY(N/O) chimeras and site-directed PVY mutants were constructed to test the involvement of different parts of the PVY genome (from nucleotide 421 to nucleotide 9629) in the induction of TVN symptoms. The analysis of both the genomic characteristics and biological properties of these mutants made it possible to highlight the involvement, in addition to residues K(400) and E(419), of the residue N(339) of the HC-Pro protein and two regions in the cytoplasmic inclusion (CI) protein to nuclear inclusion protein a-protease (NIa-Pro) sequence (nucleotides 5496-5932 and 6233-6444) in the induction of vein necrosis in tobacco infected by PVY isolates.
Project description:Potato virus Y has emerged as a threatening problem in all potato growing areas around the globe. PVY reduces the yield and quality of potato cultivars. During the last 30 years, significant genetic changes in PVY strains have been observed with an increased incidence associated with crop damage. In the current study, computational approaches were applied to predict Potato derived miRNA targets in the PVY genome. The PVY genome is approximately 9 thousand nucleotides, which transcribes the following 6 genes:CI, NIa, NIb-Pro, HC-Pro, CP, and VPg. A total of 343 mature miRNAs were retrieved from the miRBase database and were examined for their target sequences in PVY genes using the minimum free energy (mfe), minimum folding energy, sequence complementarity and mRNA-miRNA hybridization approaches. The identified potato miRNAs against viral mRNA targets have antiviral activities, leading to translational inhibition by mRNA cleavage and/or mRNA blockage. We found 86 miRNAs targeting the PVY genome at 151 different sites. Moreover, only 36 miRNAs potentially targeted the PVY genome at 101 loci. The CI gene of the PVY genome was targeted by 32 miRNAs followed by the complementarity of 26, 19, 18, 16, and 13 miRNAs. Most importantly, we found 5 miRNAs (miR160a-5p, miR7997b, miR166c-3p, miR399h, and miR5303d) that could target the CI, NIa, NIb-Pro, HC-Pro, CP, and VPg genes of PVY. The predicted miRNAs can be used for the development of PVY-resistant potato crops in the future.
Project description:In this study, the full-length nucleotide sequences of four Iranian PVY isolates belonging to PVY(N) strain were determined. The genome of Iranian PVY isolates were 9,703-9,707 nucleotides long encoding all potyviral cistrons including P1, HC-Pro, P3, 6K1, CI, 6K2, VPg, NIa-Pro, NIb and CP with coding regions of 825, 1,395, 1,095, 156, 1,902, 156, 564, 732, 1,557 and 801 nucleotides in length, respectively. The length of pipo, embedded in the P3 cistron, was 231 nucleotides. Phylogenetic analysis showed that the Iranian isolates clustered with European recombinant NTN isolates in the N lineage. Recombination analysis demonstrated that Iranian PVY(N) isolates had a typical European PVY(NTN) genome having three recombinant junctions while PVY(N) and PVY(O) were identified as the parents. We used dN/dS methods to detect candidate amino acid positions for positive selection in viral proteins. The mean ? ratio differed among different genes. Using model M0, ? values were 0.267 (P1), 0.085 (HC-Pro), 0.153 (P3), 0.050 (CI), 0.078 (VPg), 0.087 (NIa-pro), 0.079 (NIb) and 0.165 (CP). The analysis showed different sites within P1, P3 and CP were under positive selection pressure, however, the sites varied among PVY populations. To the best of our knowledge, our analysis provides the first demonstration of population structure of PVY(N) strain in mid-Eurasia Iran using complete genome sequences and highlights the importance of recombination and selection pressure in the evolution of PVY.
Project description:In the eukaryotic 26S proteasome, the 20S particle is regulated by six AAA ATPase subunits and, in archaea, by a homologous ring complex, PAN. To clarify the role of ATP in proteolysis, we studied how nucleotides bind to PAN. Although PAN has six identical subunits, it binds ATPs in pairs, and its subunits exhibit three conformational states with high, low, or no affinity for ATP. When PAN binds two ATP?S molecules or two ATP?S plus two ADP molecules, it is maximally active in binding protein substrates, associating with the 20S particle, and promoting 20S gate opening. However, binding of four ATP?S molecules reduces these functions. The 26S proteasome shows similar nucleotide dependence. These findings imply an ordered cyclical mechanism in which two ATPase subunits bind ATP simultaneously and dock into the 20S. These results can explain how these hexameric ATPases interact with and "wobble" on top of the heptameric 20S proteasome.
Project description:Potato virus Y (PVY) is one of the most economically important pathogen of potato that is present as biologically distinct strains. The virus-derived small interfering RNAs (vsiRNAs) from potato cv. Russet Burbank individually infected with PVY-N, PVY-NTN and PVY-O strains were recently characterized. Plant defense RNA-silencing mechanisms deployed against viruses produce vsiRNAs to degrade homologous viral transcripts. Based on sequence complementarity, the vsiRNAs can potentially degrade host RNA transcripts raising the prospect of vsiRNAs as pathogenicity determinants in virus-host interactions. This study investigated the global effects of PVY vsiRNAs on the host potato transcriptome.The strain-specific vsiRNAs of PVY, expressed in high copy number, were analyzed in silico for their proclivity to target potato coding and non-coding RNAs using psRobot and psRNATarget algorithms. Functional annotation of target coding transcripts was carried out to predict physiological effects of the vsiRNAs on the potato cv. Russet Burbank. The downregulation of selected target coding transcripts was further validated using qRT-PCR.The vsiRNAs derived from biologically distinct strains of PVY displayed diversity in terms of absolute number, copy number and hotspots for siRNAs on their respective genomes. The vsiRNAs populations were derived with a high frequency from 6 K1, P1 and Hc-Pro for PVY-N, P1, Hc-Pro and P3 for PVY-NTN, and P1, 3' UTR and NIa for PVY-O genomic regions. The number of vsiRNAs that displayed interaction with potato coding transcripts and number of putative coding target transcripts were comparable between PVY-N and PVY-O, and were relatively higher for PVY-NTN. The most abundant target non-coding RNA transcripts for the strain specific PVY-derived vsiRNAs were found to be MIR821, 28S rRNA,18S rRNA, snoR71, tRNA-Met and U5. Functional annotation and qRT-PCR validation suggested that the vsiRNAs target genes involved in plant hormone signaling, genetic information processing, plant-pathogen interactions, plant defense and stress response processes in potato.The findings suggested that the PVY-derived vsiRNAs could act as a pathogenicity determinant and as a counter-defense strategy to host RNA silencing in PVY-potato interactions. The broad range of host genes targeted by PVY vsiRNAs in infected potato suggests a diverse role for vsiRNAs that includes suppression of host stress responses and developmental processes. The interactome scenario is the first report on the interaction between one of the most important Potyvirus genome-derived siRNAs and the potato transcripts.
Project description:The 26S proteasome plays an essential role in regulating many cellular processes by the degradation of proteins targeted for breakdown by ubiquitin conjugation. The 26S complex is formed from the 20S core, which contains the proteolytic active sites, and 19S regulatory complexes, which bind to the 20S core to activate it and confer specificity for ubiquitinated protein substrates. We have determined the structure of the human 26S proteasome by electron microscopy and single particle analysis. In our reconstructions the crystallographic structure of each of the subunits of the 20S core can be unambiguously docked by direct recognition of each of their densities. Our results show for the first time that binding of the 19S regulatory particle results in the radial displacement of the adjacent subunits of the 20S core leading to opening of a wide channel into the proteolytic chamber. The analysis of a proteasome complex formed from one 20S core with a single 19S regulatory particle attached serve as control to our observations. We suggest locations for some of the 19S regulatory particle subunits.