Construction of arming Yarrowia lipolytica surface-displaying soybean seed coat peroxidase for use as whole-cell biocatalyst.

Whole-cell biocatalysts could be used in wide-ranging applications. In this study, a new kind of whole-cell biocatalyst was successfully constructed by genetically immobilizing soybean seed coat peroxidase (SBP) on the cell surface of Yarrowia lipolytica Po1h, using a new integrative surface display expression vector (pMIZY05). The coding sequence of SBP was optimized and chemically synthesized, then inserted into pMIZY05 to generate expression plasmid pMIZY05-oEp. A DNA fragment corresponding to SBP and selection marker expression cassettes, without bacterial sequences, was released from pMIZY05-oEp by enzyme digestion and used to transform host yeast cells. A transformant (CM11) with a high recombinant SBP activity of 1571.9 U/mL was obtained, and recombinant SBP was proved to be successfully anchored on cell surface by testing the activities of different cellular fractions. After optimization of culture conditions, the recombinant SBP activity of CM11 was increased to 4187.8 U/mL. Afterwards, biochemical properties of the recombinant SBP were determined: optimum catalytic conditions were 37.5℃ at pH 3.5, and recombinant SBP exhibited high stability during thermal or acidic treatment. Recombinant activity of cell-displayed SBP was re-examined at optimum temperature and pH, which promoted an increase up to 4432.5 U/mL. To our knowledge, this represents the highest activity ever reported for heterologous expression of SBP. This study also provides a useful strategy for heterologous expression of proteins which could be toxic to intracellular content of host cells.

Molecular characterization of a new recombinant brassica yellows virus infecting tobacco in China.

Brassica yellows virus (BrYV), prevalently distributed throughout mainland China and South Korea while triggering serious diseases in cruciferous crops, is proposed to be a new species in the genus Polerovirus within the family Luteoviridae. There are three distinct genotypes (BrYV-A, BrYV-B and BrYV-C) reported in cabbage and radish. Here, we describe a new BrYV isolate infecting tobacco plants in the field, which was named BrYV-NtabQJ. The complete genome sequence of BrYV-NtabQJ is 5741 nt in length, and 89% of the sequence shares higher sequence identities (about 90%) with different BrYV isolates. However, it possesses a quite divergent region within ORF5, which is more close to Beet western yellows virus (BWYV), Beet mild yellowing virus (BMYV) and Beet chlorosis virus (BChV). A significant recombination event was then detected among BrYV-NtabQJ, BrYV-B Beijng isolate (BrYV-BBJ) and BWYV Leonurus sibiricus isolate (BWYV-LS). It is proposed that BrYV-NtabQJ might be an interspecific recombinant between BrYV-BBJ and BWYV-LS, and the recombination might result in the successful aphid transmission of BrYV from cruciferous crops to tobacco. And it also poses new challenges for BrYV diagnosis and the vegetable production.

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

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

Development of three full-length infectious cDNA clones of distinct brassica yellows virus genotypes for agrobacterium-mediated inoculation.

Brassica yellows virus is a newly identified species in the genus of Polerovirus within the family Luteoviridae. Brassica yellows virus (BrYV) is prevalently distributed throughout Mainland China and South Korea, is an important virus infecting cruciferous crops. Based on six BrYV genomic sequences of isolates from oilseed rape, rutabaga, radish, and cabbage, three genotypes, BrYV-A, BrYV-B, and BrYV-C, exist, which mainly differ in the 5' terminal half of the genome. BrYV is an aphid-transmitted and phloem-limited virus. The use of infectious cDNA clones is an alternative means of infecting plants that allows reverse genetic studies to be performed. In this study, full-length cDNA clones of BrYV-A, recombinant BrYV5B3A, and BrYV-C were constructed under control of the cauliflower mosaic virus 35S promoter. An agrobacterium-mediated inoculation system of Nicotiana benthamiana was developed using these cDNA clones. Three days after infiltration with full-length BrYV cDNA clones, necrotic symptoms were observed in the inoculated leaves of N. benthamiana; however, no obvious symptoms appeared in the upper leaves. Reverse transcription-PCR (RT-PCR) and western blot detection of samples from the upper leaves showed that the maximum infection efficiency of BrYVs could reach 100%. The infectivity of the BrYV-A, BrYV-5B3A, and BrYV-C cDNA clones was further confirmed by northern hybridization. The system developed here will be useful for further studies of BrYV, such as host range, pathogenicity, viral gene functions, and plant-virus-vector interactions, and especially for discerning the differences among the three genotypes.

Complete genome sequence analysis identifies a new genotype of brassica yellows virus that infects cabbage and radish in China.

For brassica yellows virus (BrYV), proposed to be a member of a new polerovirus species, two clearly distinct genotypes (BrYV-A and BrYV-B) have been described. In this study, the complete nucleotide sequences of two BrYV isolates from radish and Chinese cabbage were determined. Sequence analysis suggested that these isolates represent a new genotype, referred to here as BrYV-C. The full-length sequences of the two BrYV-C isolates shared 93.4-94.8 % identity with BrYV-A and BrYV-B. Further phylogenetic analysis showed that the BrYV-C isolates formed a subgroup that was distinct from the BrYV-A and BrYV-B isolates based on all of the proteins except P5.

Amino acid sequence motifs essential for P0-mediated suppression of RNA silencing in an isolate of potato leafroll virus from Inner Mongolia.

Polerovirus P0 suppressors of host gene silencing contain a consensus F-box-like motif with Leu/Pro (L/P) requirements for suppressor activity. The Inner Mongolian Potato leafroll virus (PLRV) P0 protein (P0(PL-IM)) has an unusual F-box-like motif that contains a Trp/Gly (W/G) sequence and an additional GW/WG-like motif (G139/W140/G141) that is lacking in other P0 proteins. We used Agrobacterium infiltration-mediated RNA silencing assays to establish that P0(PL-IM) has a strong suppressor activity. Mutagenesis experiments demonstrated that the P0(PL-IM) F-box-like motif encompasses amino acids 76-LPRHLHYECLEWGLLCG THP-95, and that the suppressor activity is abolished by L76A, W87A, or G88A substitution. The suppressor activity is also weakened substantially by mutations within the G139/W140/G141 region and is eliminated by a mutation (F220R) in a C-terminal conserved sequence of P0(PL-IM). As has been observed with other P0 proteins, P0(PL-IM) suppression is correlated with reduced accumulation of the host AGO1-silencing complex protein. However, P0(PL-IM) fails to bind SKP1, which functions in a proteasome pathway that may be involved in AGO1 degradation. These results suggest that P0(PL-IM) may suppress RNA silencing by using an alternative pathway to target AGO1 for degradation. Our results help improve our understanding of the molecular mechanisms involved in PLRV infection.

Discovery and Characterization of a Novel Carlavirus Infecting Potatoes in China.

A new carlavirus, tentatively named Potato virus H (PVH), was found on potato plants with mild symptoms in Hohhot, Inner Mongolia Autonomous Region, China. PVH was confirmed by genome sequencing, serological reactions, electron microscopy, and host index assays. The PVH particles were filamentous and slightly curved, with a modal length of 570 nm. Complete RNA genomic sequences of two isolates of PVH were determined using reverse transcription-PCR (RT-PCR) and the 5' rapid amplification of cDNA ends (5' RACE) method. Sequence analysis revealed that PVH had the typical genomic organization of members of the genus Carlavirus, with a positive-sense single-stranded genome of 8410 nt. It shared coat protein (CP) and replicase amino acid sequence identities of 17.9-56.7% with those of reported carlaviruses. Phylogenetic analyses based on the protein-coding sequences of replicase and CP showed that PVH formed a distinct branch, which was related only distantly to other carlaviruses. Western blotting assays showed that PVH was not related serologically to other potato carlaviruses (Potato virus S, Potato virus M, and Potato latent virus). PVH systemically infected Nicotianaglutinosa but not Nicotiana tabacum, Nicotianabenthamiana, or Chenopodiumquinoa, which is in contrast with the other potato carlaviruses. These results support the classification of PVH as a novel species in the genus Carlavirus. Preliminary results also indicated that a cysteine-rich protein encoded by the smallest ORF located in the 3' proximal region of the genome suppressed local RNA silencing and enhanced the pathogenicity of the recombinant PVX.

Nucleotide sequence of a chickpea chlorotic stunt virus relative that infects pea and faba bean in China.

We determined the genome sequence of a new polerovirus that infects field pea and faba bean in China. Its entire nucleotide sequence (6021 nt) was most closely related (83.3% identity) to that of an Ethiopian isolate of chickpea chlorotic stunt virus (CpCSV-Eth). With the exception of the coat protein (encoded by ORF3), amino acid sequence identities of all gene products of this virus to those of CpCSV-Eth and other poleroviruses were <90%. This suggests that it is a new member of the genus Polerovirus, and the name pea mild chlorosis virus is proposed.

Complete genomic sequence analysis reveals a novel fabavirus infecting cucurbits in China.

The complete genome sequence of a cucurbit-infecting fabavirus was determined. Sequence analysis revealed that it had a genomic organization typical of fabaviruses, with genome segment sizes of 5870 nt (RNA-1) and 3294 nt (RNA-2). It shared CP and Pro-Pol amino acid sequence identities of 52.0-58.9% with those of reported fabaviruses. ELISA and western blots gave no cross-reactions between this cucurbit virus and broad bean wilt viruses 1 and 2. Based on molecular and serological criteria for species demarcation in the genus Fabavirus, the virus represents a distinct species, for which the species name Cucurbit mild mosaic virus (CuMMV) is proposed.

Molecular characterization of two genotypes of a new polerovirus infecting brassicas in China.

The genomic RNA sequences of two genotypes of a brassica-infecting polerovirus from China were determined. Sequence analysis revealed that the virus was closely related to but significantly different from turnip yellows virus (TuYV). This virus and other poleroviruses, including TuYV, had less than 90% amino acid sequence identity in all gene products except the coat protein. Based on the molecular criterion (>10% amino acid sequence difference) for species demarcation in the genus Polerovirus, the virus represents a distinct species for which the name Brassica yellows virus (BrYV) is proposed. Interestingly, there were two genotypes of BrYV, which mainly differed in the 5'-terminal half of the genome.

A novel strain of Beet western yellows virus infecting sugar beet with two distinct genotypes differing in the 5'-terminal half of genome.

The complete genomic sequences of two distinct Beet western yellows virus (BWYV) genotypes infecting sugar beet in Beijing, named as BWYV-BJ(A) and BWYV-BJ(B) (GenBank accession number HM804471, HM804472, respectively), were determined by RT-PCR sub-cloning approach. BWYV-BJ(A) and BWYV-BJ(B) were 5674 and 5626nt in length, respectively. BWYV-BJ(B) was 48nt shorter than BWYV-BJ(A) in the regions 1589-1615 and 1629-1649nt. Sequence alignment analysis showed that the full length of BWYV-BJ(A) and BWYV-BJ(B) shared 93% nucleotide sequence identity, with relatively high variability within ORFs 0, 1, 2 (at the nucleotide level was 86.3-88.8%) and high conservation within ORFs 3, 4, 5 (at the nucleotide level was 99.3-99.5%). The complete nucleotide sequences of BWYV-BJ(A) and BWYV-BJ(B) were most related to BWYV-US (80.6 and 79.0%, respectively). ORFs 1, 2 of BWYV-BJ(A) and BWYV-BJ(B) shared the highest homology with BWYV-US (nucleotide identity 91.2-93.3, 86.7-89.5%, respectively) and their ORFs 3, 4 were more closely related to BWYV-IM. However, their ORF5 were more closely related to that of Cucurbit aphid-borne yellows virus China strain (CABYV-CHN), with 68.1 and 68.5% nucleotide identity, respectively. Based on the sequence and phylogenetic analysis, we proposed that BWYV-BJ was at least a novel strain of BWYV, and BWYV-BJ(A), BWYV-BJ(B) were two distinct genotypes of BWYV-BJ. In addition, phylogenetic analysis and recombination analysis suggested that BWYV-BJ(A) and BWYV-BJ(B) might be recombinant viruses.

Ring structure amino acids affect the suppressor activity of melon aphid-borne yellows virus P0 protein.

Melon aphid-borne yellows virus (MABYV) is a newly identified polerovirus occurring in China. Here, we demonstrate that the MABYV encoded P0 (P0(MA)) protein is a strong suppressor of post-transcriptional gene silencing (PTGS) with activity comparable to tobacco etch virus (TEV) HC-Pro. In addition we have shown that the LP F-box motif present at the N-terminus of P0(MA) is required for suppressor activity. Detailed mutational analyses on P0(MA) revealed that changing the conserved Trp 212 with non-ring structured amino acids altered silencing suppressor functions. Ala substitutions at positions 12 and 211 for Phe had no effect on P0 suppression-activity, whereas Arg and Glu substitutions had greatly decreased suppressor activity. Furthermore, substitutions targeting Phe at position 30 also resulted in reduced P0 suppression-activity. Altogether, these results suggest that ring structured Trp/Phe residues in P0 have important roles in suppressor activity.

Molecular characterization of two Chinese isolates of Beet western yellows virus infecting sugar beet.

Beet western yellows virus (BWYV) has previously been reported as an agent of sugar beet yellowing disease in China. In this article, the complete genomic RNA sequences of two Chinese BWYV isolates infecting beet from Inner Mongolia (BWYV-IM) and Gansu (BWYV-GS) were determined and compared with three beet poleroviruses (BMYV, BChV and BWYV-US) and other non-beet-infecting poleroviruses. The genomes of the two isolates were 5,668 nt in length, and had almost the same genomic organization and characteristics as BWYV-US. The full length of BWYV-IM shared nucleotide sequence identities of 97.4, 86.6, 64.4 and 70.8% with BWYV-GS, BWYV-US, BChV and BMYV, respectively. Further sequence analysis indicated that the Chinese BWYV isolates were more closely related to BWYV-US; however, the identity of any gene product between the Chinese isolates and BWYV-US was <90%. Therefore, on the basis of genome sequence, we propose that these Chinese isolates are a distinct strain of BWYV that infect sugar beet. In addition, recombinant detection analysis revealed that BWYV-IM might be a recombinant virus.

Distribution and molecular diversity of three cucurbit-infecting poleroviruses in China.

Cucurbit aphid-borne yellows virus (CABYV) and Melon aphid-borne yellows virus (MABYV) have been found to be associated with cucurbit yellowing disease in China. Our report identifies for the first time a third distinct polerovirus, tentatively named Suakwa aphid-borne yellows virus (SABYV), infecting Suakwa vegetable sponge. To better understand the distribution and molecular diversity of these three poleroviruses infecting cucurbits, a total of 214 cucurbitaceous crop samples were collected from 25 provinces in China, and were investigated by RT-PCR and sequencing. Of these, 108 samples tested positive for CABYV, while 40 samples from five provinces were positive for MABYV, and SABYV was detected in only 4 samples which were collected in the southern part of China. Forty-one PCR-amplified fragments containing a portion of the RdRp gene, intergenic NCR and CP gene were cloned and sequenced. Sequence comparisons showed that CABYV isolates shared 78.0-79.2% nucleotide sequence identity with MABYV isolates, and 69.7-70.8% with SABYV. Sequence identity between MABYV and SABYV was 73.3-76.5%. In contrast, the nucleotide identities within each species were 93.2-98.7% (CABYV), 98.1-99.9% (MABYV), and 96.1-98.6% (SABYV). Phylogenetic analyses revealed that the polerovirus isolates fit into three distinct groups, corresponding to the three species. The CABYV group could be further divided into two subgroups: the Asia subgroup and the Mediterranean subgroup, based on CP gene and partial RdRp gene sequences. Recombination analysis suggested that MABYV may be a recombinant virus.

Complete sequence analysis reveals two distinct poleroviruses infecting cucurbits in China.

The complete RNA genomes of a Chinese isolate of cucurbit aphid-borne yellows virus (CABYV-CHN) and a new polerovirus tentatively referred to as melon aphid-borne yellows virus (MABYV) were determined. The entire genome of CABYV-CHN shared 89.0% nucleotide sequence identity with the French CABYV isolate. In contrast, nucleotide sequence identities between MABYV and CABYV and other poleroviruses were in the range of 50.7-74.2%, with amino acid sequence identities ranging from 24.8 to 82.9% for individual gene products. We propose that CABYV-CHN is a strain of CABYV and that MABYV is a member of a tentative distinct species within the genus Polerovirus.