Journal: Nature Communications

Article Title: Plant viruses exploit insect salivary GAPDH to modulate plant defenses

doi: 10.1038/s41467-024-51369-8

Figure Lengend Snippet: a Release of RdGAPDH from salivary glands into the saliva and subsequently into the rice plant via R. dorsalis feeding, as determined by western blot assays. Treatment with 5% sucrose solution without exposure to R. dorsalis served as the blank. NI, non-infested. b Distribution of RdGAPDH and RdRab27a in cavities and cytoplasm of salivary gland of RGDV-free or -infected R. dorsalis , as determined by immunofluorescence microscopy. The schematic illustration of type III cells of salivary glands is shown in b-I. APL, apical plasmalemma. Cv, cavity. Bars, 10 μm. c Number of RdGAPDH puncta colocalizing with RdRab27a puncta in RGDV-free or -infected salivary glands. V + , RGDV-infected R. dorsalis . V - , RGDV-free R. dorsalis . d Distribution of RdGAPDH, RdRab27a, and P8 in rice phloem exposed to RGDV-free or -infected R. dorsalis , as determined by immunofluorescence microscopy. Panels III to V (merged) are the enlarged images of the boxed areas in ( d ). P phloem, AS air space, PV pitted vessel of xylem, ST sieve tube. Bars, 10 μm. The normalized fluorescence intensity of RdGAPDH or RdRab27a in rice phloem was shown in ( d - II ). NI vs V - , p value: 0.000075. NI vs V + , p value: 0.000000000007. NI vs V - , p value: 0.00000002. NI vs V + , p value: 0.000000000004. e , f Inhibition of exosome production by RdRab27a knockdown or GW4869 treatment reduced RdGAPDH accumulation in the salivary glands of RGDV-free R. dorsalis leafhoppers and decreased RdGAPDH release into rice plants, as determined by RT-qPCR and western blot assays. The means (±SD) in ( e ) show data from the salivary glands of 30 RGDV-free R. dorsalis leafhoppers, n = 30, p value: 0.0001. g RGDV infection inducing RdGAPDH and RdRab27a expression in the salivary glands and subsequent release into the saliva and then into rice plants. SG, salivary gland. The data in ( a ), ( b ), ( d ), ( e ), ( f ), and ( g ) represent at least three biological replicates. Differences between the means (±SD) in ( c – e ) were analyzed using two-tailed unpaired t -tests. In ( a ), ( f ), and ( g ), bands for histone H3 show loading of equal amounts of protein. Source data are provided as a file.

Article Snippet: Polyclonal rabbit antibodies against RdRab27a, RdGAPDH, OsGAPDH, and RGDV P8 peptides were prepared by Genscript Biotech Corporation in Nanjing, China.

Techniques: Western Blot, Infection, Immunofluorescence, Microscopy, Fluorescence, Inhibition, Knockdown, Quantitative RT-PCR, Expressing, Two Tailed Test

Journal: Nature Communications

Article Title: Plant viruses exploit insect salivary GAPDH to modulate plant defenses

doi: 10.1038/s41467-024-51369-8

Figure Lengend Snippet: a , b Knocking down RdGAPDH expression decreased RdGAPDH accumulation in salivary glands and reduced RdGAPDH release into rice seedlings. Means (±SD) in ( a ) represent data from the salivary glands of 30 leafhoppers treated with dsRdGAPDH or dsGFP, n = 30. Proteins from the salivary glands of 20 dsRdGAPDH- or dsGFP-treated leafhoppers, as well as rice leaves exposed to these leafhoppers, were detected in western blot assays ( b ). Bands for histone H3 show loading of equal amounts of protein. SG salivary gland. c Knocking down RdGAPDH expression had a limited effect on the contents of JA and SA in rice plants, as determined by mass spectrometer assays. Means (±SD) represent data from the feeding region of rice seedlings exposed to 20 RGDV-free R. dorsalis leafhoppers for 12 h, n = 20. d Knocking down RdGAPDH expression increased H 2 O 2 burst and metabolism in rice plants, as determined by the contents of H 2 O 2 and MDA as well as POD activity. Means (±SD) represent data from the feeding region of rice seedlings exposed to 20 dsRdGAPDH- or dsGFP-treated leafhoppers, n = 20. e , f Knocking down RdGAPDH expression increased the accumulation of H 2 O 2 at feeding sites and the number of feeding sites, as determined by H2DCFDA staining. The feeding region of rice seedlings exposed to 30 dsRdGAPDH- or dsGFP-treated leafhoppers for 12 h was tested. Bars, 200 μm. Panels I and II show enlargements of the boxed areas I and II in the merged images. Means (±SD) in ( f ) show the number of feeding sites per 40 cm² of leaves, n = 30. g Knocking down RdGAPDH expression increased feeding difficulty for R. dorsalis , as determined by EPG assays. Data in ( a ), ( b ), ( c ), ( d ), ( e ), and ( f ) represent at least three biological replicates. Data in ( g ) represent 10 valid biological replicates. Differences between means (±SD) in ( c ), ( d ), ( f ), and ( g ) were analyzed using two-tailed unpaired t -tests, those in ( a ) were analyzed using two-tailed paired t -tests. Ns, not significant. Source data are provided as a file.

Article Snippet: Polyclonal rabbit antibodies against RdRab27a, RdGAPDH, OsGAPDH, and RGDV P8 peptides were prepared by Genscript Biotech Corporation in Nanjing, China.

Techniques: Expressing, Western Blot, Mass Spectrometry, Activity Assay, Staining, Two Tailed Test

Journal: Nature Communications

Article Title: Plant viruses exploit insect salivary GAPDH to modulate plant defenses

doi: 10.1038/s41467-024-51369-8

Figure Lengend Snippet: a Exposure to RGDV-infected R. dorsalis increased H 2 O 2 accumulation at the phloem of feeding sites. Panels I and II show enlargements of the boxed areas I and II in the merged images. P phloem, PV pitted vessel, ST sieve tube; V + , RGDV-infected R. dorsalis ; V - , RGDV-free R. dorsalis . Bars, 10 μm. b , c Knocking down RdGAPDH expression of R. dorsalis reduced RGDV release to rice plants. The means (±SD) in ( b ) show data from salivary glands of 30 RGDV-infected leafhoppers, n = 30, p value: 0.000006. Proteins were from the salivary glands of 20 RGDV-infected leafhoppers, as well as rice leaves exposed to these leafhoppers ( c ). Bands for histone H3 show loading of equal amounts of protein. SG salivary gland. d Knocking down RdGAPDH expression in RGDV-infected R. dorsalis enhanced H 2 O 2 burst and metabolism in rice plants. The means (±SD) show data from feeding region of rice seedlings exposed to 20 dsRNA-treated RGDV-infected leafhoppers and represent three replicates, n = 20. e Knocking down RdGAPDH expression in RGDV-infected R. dorsalis increased the number of feeding sites. Means (±SD) show the number of feeding sites per 40 cm² of leaves, n = 30. f Knockdown of RdGAPDH expression in RGDV-infected R. dorsalis hindered RGDV-infected R. dorsalis feeding, as determined by EPG assays. g Knocking down RdGAPDH expression in RGDV-infected R. dorsalis reduced the RGDV transmission rate. The means (±SD) show data from 100 dsRNA-treated RGDV-infected leafhoppers individually feeding on one new rice seedling for 24 h each day, n = 100. dsGFP vs dsRdGAPDH at 18 days padp, p value: 0.000059. h Knockdown of RdGAPDH expression decreased the survival of RGDV-infected leafhoppers. The means (±SD) show data on the survival rate of 100 dsRNA-treated RGDV-infected leafhoppers each day, n = 100. The significance of differences between dsGFP and dsRdGAPDH treatments at the same day was tested. dsGFP vs dsRdGAPDH at 17 days padp, p value: 0.00007. dsGFP vs dsRdGAPDH at 18 days padp, p value: 0.000011. The data in ( a ), ( b ), ( c ), ( d ), ( e ), ( g ), and ( h ) represent at least three biological replicates. The data in ( f ) represent 10 valid biological replicates. Differences between the means (±SD) in ( b ), ( d ), ( e ), ( f ), ( g ), and ( h ) were analyzed using two-tailed unpaired t -tests. Ns, not significant. Source data are provided as a file.

Article Snippet: Polyclonal rabbit antibodies against RdRab27a, RdGAPDH, OsGAPDH, and RGDV P8 peptides were prepared by Genscript Biotech Corporation in Nanjing, China.

Techniques: Infection, Expressing, Knockdown, Transmission Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Plant viruses exploit insect salivary GAPDH to modulate plant defenses

doi: 10.1038/s41467-024-51369-8

Figure Lengend Snippet: a Overexpression of RdGAPDH promoted RGDV release, as determined by western blot assays. Proteins were extracted from WT or RdGAODH-OE plants that were non-infested or infested with RGDV-infected R. dorsalis . Bands for histone H3 show loading of equal amounts of protein. b Overexpression of RdGAPDH reduced H 2 O 2 burst and metabolism in rice plants exposed to RGDV-free or -infected R. dorsalis , as determined by the contents of H 2 O 2 and MDA, as well as POD activity. Means (±SD) show data from the feeding region of RdGAPDH-OE plants exposed to 20 RGDV-free or -infected leafhoppers for 12 h, n = 20. V + , RGDV-infected R. dorsalis ; V - , RGDV-free R. dorsalis . c , d Exposure of RdGAPDH-OE to RGDV-free or -infected R. dorsalis reduced the accumulation of H 2 O 2 at feeding sites and the number of feeding sites, as detected by H2DCFDA staining assays. The feeding region of RdGAPDH-OE or WT seedlings exposed to 30 RGDV-free leafhoppers for 12 h was tested, n = 30. Panels I and II show enlargements of the boxed areas I and II in the merged images. Bars, 200 μm. e RdGAPDH-OE plants promoted plant penetration behavior of RGDV-free or -infected R. dorsalis , as determined by EPG assays (one-way HSD test). f RdGAPDH-OE plants increased RGDV transmission by R. dorsalis leafhoppers, as indicated by the transmission rate. Data are shown for RdGAPDH-OE #3, n (WT) = 20; n ( RdGAPDH-OE ) = 16. The data in ( a ), ( b ), ( c ), ( d ), and ( f ) represent at least three biological replicates. The data in ( e ) represent 10 valid biological replicates. Differences between the means (±SD) in ( b ), ( d ) and ( f ) were analyzed using two-tailed unpaired t -tests. Differences between the means (±SD) in ( e ) were analyzed using Tukey’s HSD test analysis. Ns, not significant. Source data are provided as a file.

Article Snippet: Polyclonal rabbit antibodies against RdRab27a, RdGAPDH, OsGAPDH, and RGDV P8 peptides were prepared by Genscript Biotech Corporation in Nanjing, China.

Techniques: Over Expression, Western Blot, Infection, Activity Assay, Staining, Transmission Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Plant viruses exploit insect salivary GAPDH to modulate plant defenses

doi: 10.1038/s41467-024-51369-8

Figure Lengend Snippet: a Schematic representation illustrating the oxidation steps of GAPDH by H 2 O 2 . b Exposure to dsRdGAPDH-treated RGDV-infected or RGDV-free R. dorsalis inhibited GAPDH oxidation in rice plants. c RdGAPDH-OE plants possessed a higher level of RdGAPDH oxidation compared to WT plants. The means (±SD) in ( b ) and ( c ) represent data from the feeding region of WT or RdGAPDH-OE #3 plants exposed to 20 leafhoppers, n = 20. V + , RGDV-infected R. dorsalis ; V - , RGDV-free R. dorsalis . d Increased H 2 O 2 enhanced RdGAPDH oxidation in vitro, as determined by the dehydrogenase activity of GAPDH, the content of –SH groups, and the acylphosphatase activity of GAPDH–SOH. Recombinant His-RdGAPDH proteins were incubated with H 2 O 2 at a series of concentrations. The significance of differences between 0 μM H 2 O 2 and other concentrations of H 2 O 2 was tested. n = 1. e Increased H 2 O 2 promoted the formation of disulfide-bonded RdGAPDH dimers in vitro. f RdGAPDHΔ failed to be oxidized by H 2 O 2 . In ( e ) and ( f ), samples derive from the same experiment and blots were processed in parallel. Equal amounts of protein were loaded onto Coomassie Brilliant Blue-stained (CBB) gels. Relative intensity of each protein band was normalized against that of the control (0 μM H 2 O 2 in ( e ) and His-RdGAPDH treatment in ( f )), and the ratios are from the corresponding blots. g Injection of rice seedlings with His-RdGAPDHΔ failed to be oxidized by H 2 O 2 in plants. n = 1. Acylphosphatase activity of GAPDH–SOH, p value: 0.000062. h Exposure of rice seedlings to RGDV-free R. dorsalis for 24 or 48 h promoted the formation of disulfide-bonded RdGAPDH dimers, as determined by western blot assays. Bands for histone H3 show loading of equal amounts of protein. i Phenotypic traits of rice seedlings exposed to leafhoppers for 48 h. Panel I shows enlarged images of the boxed area. j Cell death in rice leaves exposed to leafhoppers for 12, 24 and 48 h and in rice leaves without exposure, as determined by trypan blue staining. The blue intensity of stained rice leaves was shown in ( j-II ). NI, non-infested. The data in ( b ), ( c ), ( g ), ( h ), ( i ) and ( j ) represent at least three biological replicates, those in ( d ), ( e ), and ( f ) represent at least three technical replicates. Differences between the means (±SD) in ( b ), ( c ), ( d ), ( g ) and ( j-II ) were analyzed using two-tailed unpaired t -tests. Source data are provided as a file.

Article Snippet: Polyclonal rabbit antibodies against RdRab27a, RdGAPDH, OsGAPDH, and RGDV P8 peptides were prepared by Genscript Biotech Corporation in Nanjing, China.

Techniques: Infection, In Vitro, Activity Assay, Recombinant, Incubation, Staining, Control, Injection, Western Blot, Two Tailed Test

Journal: Nature Communications

Article Title: Plant viruses exploit insect salivary GAPDH to modulate plant defenses

doi: 10.1038/s41467-024-51369-8

Figure Lengend Snippet: a Schematic representation of GSH preventing GAPDH overoxidation through S -glutathionylation, in which the –SOH group forms a conjugate with GSH. b Exposure to RGDV-infected R. dorsalis promoted S -glutathionylation of GAPDH in rice plants. V + , RGDV-infected R. dorsalis ; V - , RGDV-free R. dorsalis . c Exposure to dsRdGAPDH-treated RGDV-free or -infected R. dorsalis reduced S -glutathionylation of GAPDH in rice plants. dsGFP vs dsRdGAPDH(V + ):–SSG group, p value: 0.000098. d RdGAPDH-OE exposed or not exposed to R. dorsalis possessed higher S -glutathionylation of GAPDH compared to WT plants. Means (±SD) in ( b – d ) show data from the feeding region exposed to 20 leafhoppers, n = 20. e Increased GSH reduced the RdGAPDH overoxidation in vitro. A mixture of His-RdGAPDH and H 2 O 2 was incubated with GSH at a series of concentrations. The significance of differences between 0 mM GSH and other concentrations of GSH treatments was tested. 0 mM vs 4.8 mM GSH: –SSG groups, p value: 0.00005. f Increased GSH reduced the formation of disulfide-bonded RdGAPDH dimers in vitro. Samples derive from the same experiment and blots were processed in parallel. Equal amounts of protein were loaded onto CBB gels. The relative intensity of each protein band was normalized against that of control (0 mM GSH), and ratios are from corresponding blots. g Increased H 2 O 2 reduced GSH S -glutathionylation. A mixture of His-RdGAPDH and GSH was incubated with H 2 O 2 at a series of concentrations. The significance of differences between 0 μM H 2 O 2 and other concentrations of H 2 O 2 treatments was tested. –SSG groups: 0 μM vs 1 μM H 2 O 2 , p value: 0.000058; 0 μM vs 500 μM H 2 O 2 , p value: 0.000043. h Injection of rice seedlings with His-RdGAPDH induced a higher content of GSH compared to His-RdGAPDHΔ treatment. n = 1. i BSO treatment reduced the S -glutathionylation of GAPDH but increased acylphosphatase activity of GAPDH–SOH. The stem bases of rice seedlings were injected with BSO or H 2 O and then exposed to 20 RGDV-free leafhoppers. n = 20. j Exposure of BSO-treated rice seedlings to R. dorsalis promoted the formation of disulfide-bonded RdGAPDH dimers in rice seedlings. Bands for histone H3 show loading of equal amounts of protein. The data in ( b ), ( c ), ( d ), ( h ), ( i ), and ( j ) represent at least three biological replicates. The data in ( e ), ( f ) and ( g ) represent three technical replicates. Differences between the means (±SD) in ( b ), ( c ), ( d ), ( e ), ( g ), ( h ), and ( i ) were analyzed using two-tailed unpaired t -tests. Source data are provided as a file.

Article Snippet: Polyclonal rabbit antibodies against RdRab27a, RdGAPDH, OsGAPDH, and RGDV P8 peptides were prepared by Genscript Biotech Corporation in Nanjing, China.

Techniques: Infection, In Vitro, Incubation, Control, Injection, Activity Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Plant viruses exploit insect salivary GAPDH to modulate plant defenses

doi: 10.1038/s41467-024-51369-8

Figure Lengend Snippet: Under the proposed model, RdGAPDH in salivary glands is loaded into exosomes for release into cavities and ultimately enters into the rice phloem via saliva during R. dorsalis feeding. The released RdGAPDH is oxidized by H 2 O 2 in the rice phloem, leading to a decrease in H 2 O 2 burst, thus benefiting R. dorsalis feeding. However, overoxidation of RdGAPDH by H 2 O 2 irreversibly generates toxic disulfide-bonded dimers. GSH in rice cells prevents RdGAPDH overoxidation through S -glutathionylation, thereby reducing the toxicity of the overoxidized RdGAPDH. RGDV infection promotes RdGAPDH to be loaded into exosomes for release into the rice phloem. The increase in RdGAPDH enhances decreases in H 2 O 2 levels, thus promoting viral transmission. Induced S -glutathionylation of RdGAPDH also decreases RdGAPDH-induced oxidative stress, ultimately achieving a balance among the virus, leafhopper, and rice.

Article Snippet: Polyclonal rabbit antibodies against RdRab27a, RdGAPDH, OsGAPDH, and RGDV P8 peptides were prepared by Genscript Biotech Corporation in Nanjing, China.

Techniques: Infection, Transmission Assay, Virus

Journal: PLoS Genetics

Article Title: Chromosome separation during Drosophila male meiosis I requires separase-mediated cleavage of the homolog conjunction protein UNO

doi: 10.1371/journal.pgen.1008928

Figure Lengend Snippet: (A) Testis mass isolation. After grinding up third instar wandering stage larvae and early pupae with EGFP expression in testes, sieving and density gradient centrifugation were used for enrichment of testes. Fluorescent particle sorting was applied as the final purification step. The procedure allowed an isolation of around five thousand testes per run. For each of the three genotypes (bam>mnm-EGFP, bam>snm-EGFP, and exuM-EGFP) around 30’000 testes were prepared for four replicates of affinity-purification with anti-EGFP beads. (B) VENN diagram summarizing the numbers of co-purified proteins identified by mass spectrometry after affinity-purification of MNM-EGFP, SNM-EGFP or EGFP from testes extracts. (C) Bar diagram presenting the spectral counts of peptides derived from MNM, SNM, TEF and UNO that were detected in each of the four replicate samples obtained by affinity-purification of MNM-EGFP, SNM-EGFP and EGFP from testis extracts. (D) The function of univalents only ( uno ), a gene coding for a protein co-purified with MNM-EGFP and SNM-EGFP, was further characterized. Four distinct, intragenic out-of-frame deletions (cc1 –cc4) generated by CRISPR/cas eliminate parts of the coding region (black box) as indicated approximately by the red box. The green bar indicates the genomic region present in the g-uno transgenes, which had either an N-terminal, a C-terminal, or no in-frame fusion to the EGFP coding sequence. (E) Amino acid sequence conservation in UNO. Drosophila melanogaster UNO contains 417 amino acids. Positions and extent of amino acid identity observed in different regions based on an alignment of drosophilid UNO orthologs are indicated. Two regions with stronger sequence conservation close to the N- and C-termini (yellow and blue, respectively) are connected by a poorly conserved linker region, which includes a well-conserved match to the separase cleavage site consensus (grey).

Article Snippet: Immunolabeling of UNO was done with affinity-purified rabbit polyclonal antibodies (rb34) against a C-terminal UNO peptide (MERETHRRWSAVPPEGNPV; Moravian Biotechnology, Brno, Czech Republic) at 1:1000.

Techniques: Isolation, Expressing, Gradient Centrifugation, Purification, Affinity Purification, Mass Spectrometry, Derivative Assay, Generated, CRISPR, Sequencing

Journal: PLoS Genetics

Article Title: Chromosome separation during Drosophila male meiosis I requires separase-mediated cleavage of the homolog conjunction protein UNO

doi: 10.1371/journal.pgen.1008928

Figure Lengend Snippet: (A,B) Immunolabeling with anti-UNO. (A) Whole mount preparations of testes dissected from either control (+) or uno cc1 hemizygous males ( uno cc1 / Df ) were labeled with anti-UNO and anti-Lamin. (B) Squash preparations of control testes were labeled with anti-UNO, anti-Lamin and a DNA stain. High magnification views of cells at the indicated stages are shown. In S5 spermatocytes (S5), anti-UNO signals are highest in several subnucleolar foci. In prometaphase I (prometa I), after chromosome condensation, these appear to have coalesced into a single bright dot on the sex chromosome bivalent (arrow). Postmeiotic spermatids (spermatid) do not display any specific anti-UNO signals. (C) UNO-EGFP signals. Squash preparations were made with testes dissected from uno null mutants expressing UNO-EGFP from a transgene under control of the uno cis-regulatory region ( uno cc1 / Df(2R)Exel7094 ; g-uno-EGFP III . 1/+ ). High magnification views of cells at the indicated stages are shown. In S5 spermatocytes (S5), UNO-EGFP signals are maximal in subnucleolar foci. After adjusting display settings to reveal low intensity EGFP signals (lower panel), fine dot signals are also apparent on autosomal chromatin (arrows). Similarly, in prometaphase I (prometa I) a single bright UNO-EGFP dot marks the sex chromosome bivalent, while far weaker dots are apparent on autosomal bivalents (lower panel, arrows). Scale bars = 25 μm (A) and 5 μm (B,C).

Article Snippet: Immunolabeling of UNO was done with affinity-purified rabbit polyclonal antibodies (rb34) against a C-terminal UNO peptide (MERETHRRWSAVPPEGNPV; Moravian Biotechnology, Brno, Czech Republic) at 1:1000.

Techniques: Immunolabeling, Labeling, Staining, Expressing

Journal: PLoS Genetics

Article Title: Chromosome separation during Drosophila male meiosis I requires separase-mediated cleavage of the homolog conjunction protein UNO

doi: 10.1371/journal.pgen.1008928

Figure Lengend Snippet: (A,B) Squash preparations of testes from either (A) bam>mnm-EGFP or (B) bam>snm-EGFP males were labeled with anti-UNO and a DNA stain. High magnification views of cells at the indicated stages are displayed. As further illustrated with insets, co-localization of UNO with MNM-EGFP and SNM-EGFP is evident during S5 in subnucleolar foci (nu) and during prometaphase I in a dot associated with the sex chromosome bivalent (xy). In those S5 cells, which had detectable dot-like signals within autosomal territories (a), UNO and SNM-EGFP were also largely co-localized. (C) Localization of UNO-EGFP expressed from a transgene under control of the uno cis-regulatory region ( g-uno-EGFP ) was analyzed in control ( + ) as well as in mnm (mnm - ) , snm ( snm - ) and tef ( tef - ) mutant testes. Testis tips are displayed in the four panels on the left side and high magnification views of single cells at the indicated stages on the right side. The number of UNO-EGFP dot signals in autosomal chromosome territories of S5 spermatocytes detected in control and tef mutants is presented in a histogram. (D,E) Localization of (D) MNM-EGFP and (E) SNM-EGFP in testes without ( + ) or with spermatocyte-specific UNO depletion ( uno RNAi ). The bamP-GAL4-VP16 driver in combination with UASt transgenes was used for expression of MNM-EGFP, SNM-EGFP and UNO depletion. Testis tips after a merge of the DNA and EGFP signals are displayed. The regions below the dashed white lines are also shown in the bottom row in order to document the lower EGFP signals in more mature spermatocytes, in (E) after display adjustment for further enhancement of weak signals. Scale bars = 5 μm (A, B, and C in right panels) and 50 μm (D, E and C left panels).

Article Snippet: Immunolabeling of UNO was done with affinity-purified rabbit polyclonal antibodies (rb34) against a C-terminal UNO peptide (MERETHRRWSAVPPEGNPV; Moravian Biotechnology, Brno, Czech Republic) at 1:1000.

Techniques: Labeling, Staining, Mutagenesis, Expressing

Journal: PLoS Pathogens

Article Title: A plant reovirus hijacks endoplasmic reticulum-associated degradation machinery to promote efficient viral transmission by its planthopper vector under high temperature conditions

doi: 10.1371/journal.ppat.1009347

Figure Lengend Snippet: (A) Structural features and domains of DnaJB11 and BAP31 from S . furcifera . (B) Interactions among P7-1 of SRBSDV, BAP31 and DnaJB11 were detected by yeast two-hybrid assay. Transformants on plate of QDO culture medium were labeled as follows: P7-1+DnaJB11N, pGBKT7-P7-1/pGADT7-DnaJB11N; P7-1+DnaJB11C, pGBKT7-P7-1/pGADT7-DnaJB11C; P7-1+BAP31, pGBKT7-P7-1/pGADT7-BAP31; BAP31+DnaJB11N, pGBKT7-BAP31/pGADT7-DnaJB11N; BAP31+DnaJB11C, pGBKT7-BAP31/pGADT7-DnaJB11C; Positive control, pGBKT7-53/pGADT7-T; Negative control, pGBKT7-Lam/pGADT7-T. Serially diluted yeast cultures were shown. (C) Both DnaJB11C and BAP31 interacted with the TM1-containing N-terminus, but not with the TM2-containing C-terminus of SRBSDV P7-1, as detected by yeast two-hybrid assay. Transformants on plate of DDO or QDO+X-α-Gal culture medium. (D-G) The interactions among P7-1, BAP31 and DnaJB11C were demonstrated by Co-IP assay. (D) The lysate was immunoprecipitated with P7-1 antibodies, and the immunoprecipitated proteins were detected by BAP31 antibodies. (E) The lysate was immunoprecipitated with P7-1 antibodies, and the immunoprecipitated proteins were detected by DnaJB11 antibodies. (F) The lysate was immunoprecipitated with BAP31 antibodies, and the immunoprecipitated proteins were detected by DnaJB11 antibodies. (G) The lysate was immunoprecipitated with P7-1 antibodies, and the immunoprecipitated proteins were detected by GFP antibodies. (H, I) The competitive interactions among P7-1 of SRBSDV, BAP31 and DnaJB11C were detected by pull-down assay. P7-1-His and MBP-BAP31 were incubated with Ni-NTA agarose beads, then GST-DnaJB11C was added to the beads; when increased the amounts of DnaJB11C, the binding between P7-1 and BAP31 was decreased (H). P7-1-His and GST-DnaJB11C were incubated with Ni-NTA agarose beads, then MBP-BAP31 was added to the beads; when increased the amounts of MBP-BAP31, the binding between P7-1 and DnaJB11C was not affected (I).

Article Snippet: Polyclonal antibodies against DnaJB11 peptide ARIRKKNEGMPVYN was prepared in rabbit by Genscript USA Innovation Company (Nanjing), which is approved by the Science Technology Department of Jiangsu Province, China with approval number SYXK (Su) 2018–0015.

Techniques: Y2H Assay, Labeling, Positive Control, Negative Control, Co-Immunoprecipitation Assay, Immunoprecipitation, Pull Down Assay, Incubation, Binding Assay

Journal: PLoS Pathogens

Article Title: A plant reovirus hijacks endoplasmic reticulum-associated degradation machinery to promote efficient viral transmission by its planthopper vector under high temperature conditions

doi: 10.1371/journal.ppat.1009347

Figure Lengend Snippet: (A) Sf9 cells were fixed at 18, 24, 36, or 48 hpi and immunolabeled with P7-1-FITC (green). (B) After staining with the ER-Tracker Dyes (red) for 30 min, Sf9 cells were fixed at 18 or 36 hpi and immunolabeled with P7-1-FITC (green). (C) After the incubation with 500, 250, or 125 ng/mL BFA or DMSO, Sf9 cells were fixed at 36 hpi and immunolabeled with P7-1-FITC (green). (D) After staining with the ER-Tracker Dyes (red) for 30 min, 500 ng/mL BFA-treated Sf9 cells were fixed at 36 hpi and immunolabeled with P7-1-FITC (green). (E) After staining with the ER-Tracker Dyes (red) for 30 min, Sf9 cells expressed with P7-1N or P7-1C were fixed at 36 hpi and immunolabeled with P7-1-FITC (green). (F) At 48 hpi. Sf9 cells expressed with BAP31 or DnaJB11C were fixed and immunolabeled with BAP31-FITC (green) or DnaJB11-FITC (green), respectively. (G) At 48 hpi, Sf9 cells co-expressed with BAP31 and DnaJB11C were stained with the ER-Tracker Dyes (red), and then fixed and immunolabeled with BAP31-FITC (green) and DnaJB11-specifc IgG conjugated to Alexa Fluor 647 (DnaJB11-Alexa Fluor 647, blue). (H) At 18 or 48 hpi, Sf9 cells co-expressed with BAP31 and P7-1 were fixed and immunolabeled with BAP31-FITC (green) and P7-1-rhodamine (red). (I) At 18 or 48 hpi, Sf9 cells co-expressed with P7-1 and DnaJB11C or the complete DnaJB11 were fixed and immunolabeled with P7-1-FITC (green) and DnaJB11-specific IgG conjugated to rhodamine (DnaJB11-rhodamine, red). (J) Immunogold labeling of DnaJB11 on P7-1 tubules in Sf9 cells co-expressed with P7-1 and DnaJB11C. Cells expressed with DnaJB11 alone (I) or co-expressed with P7-1 and DnaJB11C (II) were immunolabeled with DnaJB11 antibodies and goat antibodies against rabbit IgG that had been conjugated with 10-nm-diameter gold particles (arrows) as secondary antibodies. (K) At 48 hpi, Sf9 cells triply-expressed with P7-1, BAP31 and DnaJB11C were fixed and immunolabeled with BAP31-FITC (green), P7-1-rhodamine (red) and DnaJB11-Alexa Fluor 647 (blue). BF, brightfield. Bars in A-I, K, 5 μm. Bar in J, 100 nm.

Article Snippet: Polyclonal antibodies against DnaJB11 peptide ARIRKKNEGMPVYN was prepared in rabbit by Genscript USA Innovation Company (Nanjing), which is approved by the Science Technology Department of Jiangsu Province, China with approval number SYXK (Su) 2018–0015.

Techniques: Immunolabeling, Staining, Incubation, Labeling

Journal: PLoS Pathogens

Article Title: A plant reovirus hijacks endoplasmic reticulum-associated degradation machinery to promote efficient viral transmission by its planthopper vector under high temperature conditions

doi: 10.1371/journal.ppat.1009347

Figure Lengend Snippet: (A-B) The transcript levels of Sf-DnaJB11 (A) and Sf-BAP31 (B) in Sf9 cells infected with recombinant baculovirus containing P7-1 after treatment with dsGFP, dsSf-BAP31 or dsSf-DnaJB11 were detected by RT-qPCR assay. Means (±SD) from three biological replicates are shown. Different letters in the same column indicate a significant difference ( P <0.05, Tukey’s HSD multiple test) in Sf-BAP31 or Sf-DnaJB11 transcript levels among S . furcifera treated with different dsRNAs. (C) Sf9 cells infected with recombinant baculovirus containing P7-1 after treatment with dsGFP, dsSf-BAP31 or dsSf-DnaJB11 were fixed at 18 or 36 hpi and immunolabeled with P7-1-FITC (green). BF, brightfield. Bars, 5 μm.

Article Snippet: Polyclonal antibodies against DnaJB11 peptide ARIRKKNEGMPVYN was prepared in rabbit by Genscript USA Innovation Company (Nanjing), which is approved by the Science Technology Department of Jiangsu Province, China with approval number SYXK (Su) 2018–0015.

Techniques: Infection, Recombinant, Quantitative RT-PCR, Immunolabeling

Journal: PLoS Pathogens

Article Title: A plant reovirus hijacks endoplasmic reticulum-associated degradation machinery to promote efficient viral transmission by its planthopper vector under high temperature conditions

doi: 10.1371/journal.ppat.1009347

Figure Lengend Snippet: (A) Effects of SRBSDV infection on the transcript levels of BAP31 and DnaJB11 in viruliferous or nonviruliferous insects, as detected by RT-qPCR assay. Error bars indicate standard deviations from three biological replicates. Means (±SD) from three biological replicates are shown. Different letters in the same column indicate a significant difference ( P <0.05, Tukey’s HSD multiple test) in BAP31 or DnaJB11 transcript levels between nonviruliferous and viruliferous S . furcifera . (B) Effects of SRBSDV infection on the protein levels of BAP31, DnaJB11, P9-1 or P7-1 in viruliferous or nonviruliferous insects, as detected by western blot assay by using BAP31-, DnaJB11-, P9-1- or P7-1-specific IgGs. Insect GAPDH was used as an internal control. (C) At 6 days padp, the midguts of nonviruliferous or viruliferous insects were immunolabeled with DnaJB11-FITC (green) and P7-1-rhodamine (red) and then examined by immunofluorescence microscopy. DnaJB11 and P7-1 tubules were co-localized in the midgut. mg, midgut. Bars, 30 μm. (D) Immunogold labeling of DnaJB11 on virus-containing tubules in SRBSDV-infected midgut from nonviruliferous (I) and viruliferous (II) insects. The midguts were immunolabled with DnaJB11 antibodies and goat antibodies against rabbit IgG that had been conjugated with 10-nm-diameter gold particles (arrows) as secondary antibodies. Bars, 100 nm. (E) At 6 days padp, the midguts of nonviruliferous or viruliferous insects were immunolabeled with BAP31-FITC (green) and P7-1-rhodamine (red) and then examined by immunofluorescence microscopy. Bars, 30 μm.

Article Snippet: Polyclonal antibodies against DnaJB11 peptide ARIRKKNEGMPVYN was prepared in rabbit by Genscript USA Innovation Company (Nanjing), which is approved by the Science Technology Department of Jiangsu Province, China with approval number SYXK (Su) 2018–0015.

Techniques: Infection, Quantitative RT-PCR, Western Blot, Control, Immunolabeling, Immunofluorescence, Microscopy, Labeling, Virus

Journal: PLoS Pathogens

Article Title: A plant reovirus hijacks endoplasmic reticulum-associated degradation machinery to promote efficient viral transmission by its planthopper vector under high temperature conditions

doi: 10.1371/journal.ppat.1009347

Figure Lengend Snippet: (A-D) The transcript levels of BAP31 (A), DnaJB11 (B), P9-1 (C), or P7-1 (D) from dsRNAs (dsGFP, dsBAP31 and dsDnaJB11)-treated or untreated viruliferous insects were detected by RT-qPCR assay. Means (±SD) from three biological replicates are shown. Different letters in the same column indicate a significant difference ( P <0.05, Tukey’s HSD multiple test) in BAP31, DnaJB11, P9-1 or P7-1 transcript levels among viruliferous S . furcifera treated with different dsRNAs. (E) The protein levels of P9-1, P7-1, DnaJB11 or BAP31 from dsRNAs-treated virulifeorus insects were detected by western blot assay by using P9-1-, P7-1-, DnaJB11-, or BAP31-specific IgGs. Insect GAPDH was used as an internal control. (F) The visceral muscles of the midguts of viruliferous dsRNAs-treated and untreated insects were immunolabeled with P9-1-FITC (green), P7-1-rhodamine (red) and the actin dye phalloidin-Alexa Fluor 647 (blue), then examined by immunofluorescence microscopy. vm, visceral muscles. Bars, 30 μm. (G) Transmission rates of SRBSDV by individual viruliferous S . furcifera untreated or treated with different dsRNAs. Means (±SD) from three biological replicates are shown. Different letters in the same column indicate a significant difference ( P <0.05, Tukey’s HSD multiple test) in transmission rates of SRBSDV by different dsRNAs-treated viruliferous S . furcifera . NT, untreated viruliferous insects.

Article Snippet: Polyclonal antibodies against DnaJB11 peptide ARIRKKNEGMPVYN was prepared in rabbit by Genscript USA Innovation Company (Nanjing), which is approved by the Science Technology Department of Jiangsu Province, China with approval number SYXK (Su) 2018–0015.

Techniques: Quantitative RT-PCR, Western Blot, Control, Muscles, Immunolabeling, Immunofluorescence, Microscopy, Transmission Assay

Journal: PLoS Pathogens

Article Title: A plant reovirus hijacks endoplasmic reticulum-associated degradation machinery to promote efficient viral transmission by its planthopper vector under high temperature conditions

doi: 10.1371/journal.ppat.1009347

Figure Lengend Snippet: (A-B) The transcript levels of DnaJB11 (A) and BAP31 (B) in nonviruliferous or viruliferous insects after incubation with 15, 20, 25 and 35°C at 6 days padp were measured by RT-qPCR assay. Error bars indicate standard deviations from three biological replicates. Means (±SD) from three biological replicates are shown. Multiple comparisons of the means were conducted using a Tukey’s honest significant difference (HSD) test with a one-way analysis of variance (ANOVA). * P <0.05, ** P <0.01. (C) The protein levels of BAP31 or DnaJB11 in viruliferous insects after incubation with 15, 20, 25 and 35°C at 6 days padp were detected by western blot assay by using BAP31- or DnaJB11-specific IgGs. Insect GAPDH was detected with GAPDH-specific IgG as an internal control.

Article Snippet: Polyclonal antibodies against DnaJB11 peptide ARIRKKNEGMPVYN was prepared in rabbit by Genscript USA Innovation Company (Nanjing), which is approved by the Science Technology Department of Jiangsu Province, China with approval number SYXK (Su) 2018–0015.

Techniques: Incubation, Quantitative RT-PCR, Western Blot, Control

Journal: PLoS Pathogens

Article Title: A plant reovirus hijacks endoplasmic reticulum-associated degradation machinery to promote efficient viral transmission by its planthopper vector under high temperature conditions

doi: 10.1371/journal.ppat.1009347

Figure Lengend Snippet: Two factors of ERAD machinery, DnaJB11 and BAP31 compete to interact with the tubule protein P7-1 of SRBSDV; however, DnaJB11 promotes whereas BAP31 inhibits P7-1 tubule assembly. Furthermore, BAP31 negatively regulates DnaJB11 expression through their direct interaction. Thus, high temperature can significantly upregulate DnaJB11 expression but inhibit BAP31 expression, finally facilitating the assembly of abundant P7-1 tubules.

Article Snippet: Polyclonal antibodies against DnaJB11 peptide ARIRKKNEGMPVYN was prepared in rabbit by Genscript USA Innovation Company (Nanjing), which is approved by the Science Technology Department of Jiangsu Province, China with approval number SYXK (Su) 2018–0015.

Techniques: Expressing