Data_Sheet_1.docx

<p>The tomato resistance gene Tm-2² encodes a coiled coil-nucleotide binding site-leucine rich repeat type resistance protein and confers effective immune response against tobamoviruses by detecting the presence of viral movement proteins (MPs). In this study, we show that the Nicotiana benthamiana Heat shock protein 90-kD (Hsp90) interacts with Tm-2². Silencing of Hsp90 reduced Tm-2²-mediated resistance to Tobacco mosaic virus (TMV) and the steady-state levels of Tm-2² protein. Further, Hsp90 associates with SGT1 in yeast and in plant cells. These results suggest that Hsp90-SGT1 complex takes part in Tm-2²-mediated TMV resistance by functioning as chaperone to regulate Tm-2² stability.</p

Table_1_Methylation level of potato gene OMT30376 regulates tuber anthocyanin transformations.docx

After anthocyanin synthesis, a variety of anthocyanin compounds are produced through further methylation, glycosylation, and acylation. However, the effect of the potato methylase gene on anthocyanin biosynthesis has not been reported. Red and purple mutation types appear in tubers of the potato cultivar ‘Purple Viking’ with chimeric skin phenotypes. In this study, transcriptome and anthocyanin metabolome analyses were performed on skin of Purple Viking tubers and associated mutants. According to the metabolome analysis, the transformation of delphinidin into malvidin-3-O-glucoside and petunidin 3-O-glucoside and that of cyanidin into rosinidin O-hexoside and peonidin-3-O-glucoside were hindered in red tubers. Expression of methyltransferase gene OMT30376 was significantly lower in red tubers than in purple ones, whereas the methylation level of OMT30376 was significantly higher in red tubers. In addition, red skin appeared in tubers from purple tuber plants treated with S-adenosylmethionine (SAM), indicating the difference between purple and red was caused by the methylation degree of the gene OMT30376. Thus, the results of the study suggest that the OMT30376 gene is involved in the transformation of anthocyanins in potato tubers. The results also provide an important reference to reveal the regulatory mechanisms of anthocyanin biosynthesis and transformation.</p

Table_2_Methylation level of potato gene OMT30376 regulates tuber anthocyanin transformations.docx

After anthocyanin synthesis, a variety of anthocyanin compounds are produced through further methylation, glycosylation, and acylation. However, the effect of the potato methylase gene on anthocyanin biosynthesis has not been reported. Red and purple mutation types appear in tubers of the potato cultivar ‘Purple Viking’ with chimeric skin phenotypes. In this study, transcriptome and anthocyanin metabolome analyses were performed on skin of Purple Viking tubers and associated mutants. According to the metabolome analysis, the transformation of delphinidin into malvidin-3-O-glucoside and petunidin 3-O-glucoside and that of cyanidin into rosinidin O-hexoside and peonidin-3-O-glucoside were hindered in red tubers. Expression of methyltransferase gene OMT30376 was significantly lower in red tubers than in purple ones, whereas the methylation level of OMT30376 was significantly higher in red tubers. In addition, red skin appeared in tubers from purple tuber plants treated with S-adenosylmethionine (SAM), indicating the difference between purple and red was caused by the methylation degree of the gene OMT30376. Thus, the results of the study suggest that the OMT30376 gene is involved in the transformation of anthocyanins in potato tubers. The results also provide an important reference to reveal the regulatory mechanisms of anthocyanin biosynthesis and transformation.</p

Table_3_Methylation level of potato gene OMT30376 regulates tuber anthocyanin transformations.xlsx

After anthocyanin synthesis, a variety of anthocyanin compounds are produced through further methylation, glycosylation, and acylation. However, the effect of the potato methylase gene on anthocyanin biosynthesis has not been reported. Red and purple mutation types appear in tubers of the potato cultivar ‘Purple Viking’ with chimeric skin phenotypes. In this study, transcriptome and anthocyanin metabolome analyses were performed on skin of Purple Viking tubers and associated mutants. According to the metabolome analysis, the transformation of delphinidin into malvidin-3-O-glucoside and petunidin 3-O-glucoside and that of cyanidin into rosinidin O-hexoside and peonidin-3-O-glucoside were hindered in red tubers. Expression of methyltransferase gene OMT30376 was significantly lower in red tubers than in purple ones, whereas the methylation level of OMT30376 was significantly higher in red tubers. In addition, red skin appeared in tubers from purple tuber plants treated with S-adenosylmethionine (SAM), indicating the difference between purple and red was caused by the methylation degree of the gene OMT30376. Thus, the results of the study suggest that the OMT30376 gene is involved in the transformation of anthocyanins in potato tubers. The results also provide an important reference to reveal the regulatory mechanisms of anthocyanin biosynthesis and transformation.</p

CLCuMuB βC1 Subverts Ubiquitination by Interacting with NbSKP1s to Enhance Geminivirus Infection in <i>Nicotiana benthamiana</i>

<div><p>Viruses interfere with and usurp host machinery and circumvent defense responses to create a suitable cellular environment for successful infection. This is usually achieved through interactions between viral proteins and host factors. Geminiviruses are a group of plant-infecting DNA viruses, of which some contain a betasatellite, known as DNAβ. Here, we report that <i>Cotton leaf curl Multan virus</i> (CLCuMuV) uses its sole satellite-encoded protein βC1 to regulate the plant ubiquitination pathway for effective infection. We found that <i>CLCuMu betasatellite</i> (CLCuMuB) βC1 interacts with NbSKP1, and interrupts the interaction of NbSKP1s with NbCUL1. Silencing of either <i>NbSKP1s</i> or <i>NbCUL1</i> enhances the accumulation of CLCuMuV genomic DNA and results in severe disease symptoms in plants. βC1 impairs the integrity of SCF^COI1 and the stabilization of GAI, a substrate of the SCF^SYL1 to hinder responses to jasmonates (JA) and gibberellins (GA). Moreover, JA treatment reduces viral accumulation and symptoms. These results suggest that CLCuMuB βC1 inhibits the ubiquitination function of SCF E3 ligases through interacting with NbSKP1s to enhance CLCuMuV infection and symptom induction in plants.</p></div

The N-terminal domain of NbSKP1.1 interacts with CLCuMuB βC1 in yeast.

<p>Growth of SKY48 yeast strains containing NLS-LexA BD-CLCuMuB βC1 (BD-βC1) transformed with AD fused full length, N-terminal fragment (N98aa), C-terminal fragment (C57aa) of NbSKP1.1 or AD (control) on Leu-containing (Leu⁺) and Leu-deficient (Leu⁻) medium with galactose (Gal) and raffinose (Raf) at 28°C for 6 d. Yeast cells were plated at OD₆₀₀ = 1, 0.1, 0.01.</p

CLCuMuB βC1 hinders the degradation of YFP-GAI <i>in vivo</i>.

<p>(A) CLCuMuB βC1 attenued degradation of YFP-GAI <i>in vivo</i>. YFP-GAI expression construct was coinfiltrated with constructs expressing HA-nLUC or HA-βC1 into seven to eight-week-old <i>N</i>. <i>benthamiana</i> plant leaves. Around 48 hpi, agroinfiltrated leaves were sprayed with 100 μM GA₃ or mock solution (ethonal) and visualized via a Zeiss LSM 710 laser scanning microscope. Bar scales represents 200 μm. DMSO and MG132 (50 μM) were applied into plant leaves 12 h before observation. Protein level was analyzed via SDS-PAGE and western blot analysis with the anti-GFP antibody, which also recognizes YFP. The PVDF membrane was stained with Ponceaux to visualize the large subunit of ribulose-1,5-bisphosphate as a loading control. (B) Real-time RT-PCR detected the mRNA level of YFP-GAI. Total RNA was extracted from each <i>N</i>. <i>benthamiana</i> leaves and then subjected to quantitative RT-PCR (means±SEM, n = 3) to quantify YFP-GAI mRNA level. <i>eIF4a</i> was used as the internal reference. (C) CLCuMuB βC1 didn’t affect stability of GFP <i>in vivo</i>. Detection of GFP (as an internal control) in <i>N</i>. <i>benthamiana</i> leaves coinfiltrated with the construct expressing GFP together with constructs expressing HA-nLUC or HA-βC1 and treated with 100 μM GA₃ or mock (ethanol) solution and visualized via a Zeiss LSM 710 laser scanning microscope. Bar scale represents 200 μm. Protein level was analyzed via SDS-PAGE and immunoblot analysis with anti-GFP. The PVDF membrane was stained with Ponceaux to visualize the large subunit of ribulose-1,5-bisphosphate as a loading control.</p

Silencing of <i>NbSKP1s</i> enhances CLCuMuV DNA accumulation and results in typical disease symptoms.

<p>(A1, A2 and A3) Six- to seven-week-old <i>N</i>. <i>benthamiana</i> plants were agroinoculated with CLCuMuV (CA) and βM2-<i>SKP1</i>F1 (A1), βM2-<i>SKP1</i>F2 (A2), βM2-<i>SKP1</i>F3 (A3) or βM2-<i>βC1</i>F (as the control). (B1, B2 and B3) Silencing of <i>NbSKP1s</i> enhanced CLCuMuV DNA accumulation. Each group contained 7 plants. At 14 dpi, total DNA was extracted from each plant respectively and subjected to quantitative real-time PCR (means±SEM, n = 7) to quantify viral DNA accumulation. The internal reference method was used to calculate the relative amount of viral DNA. (C1, C2 and C3) Real-time RT-PCR confirmed silencing of <i>NbSKP1s</i>. Total RNA was extracted from each plant respectively and subjected to quantitative RT-PCR (means±SEM, n = 4) to quantify <i>NbSKP1s</i> mRNA level. <i>Actin</i> was used as the internal reference. The raw data of (B1–B3) and (C1–C3) were analysed by two-sample <i>t</i>-test to show the significance level at 0.05 (*) and 0.01 (**). These experiments were repeated at least twice. (D1, D2 and D3) 50% plants infected with CA+βM2-<i>SKP1</i>F1 (D1), 50% plants infected with CA+βM2-<i>SKP1</i>F2 (D2) and 100% plants infected with CA+βM2-<i>SKP1</i>F3 (D3) show severe symptoms at 21 dpi. (E1, E2, E3 and E4) Apical leaves of plants infected with CA+βM2-<i>βC1</i>F (E1), CA+βM2-<i>SKP1</i>F1(E2), CA+βM2-<i>SKP1</i>F2 (E3) and CA+βM2-<i>SKP1</i>F3 (E4) at 21 dpi.</p

CLCuMuB βC1 interferes with the interaction between NbCUL1 and NbSKP1.1 <i>in vitro</i> and <i>in vivo</i>.

<p>(A) GFP competitive pull-down assay <i>in vitro</i>. His-βC1 was expressed in <i>E</i>. <i>coli</i> as inclusion body and refolded through urea-arginine dialysis. BSA (NEB, USA) was used as a control. GFP-NbCUL1 or GFP was expressed in <i>N</i>. <i>benthamiana</i> leaves and trapped through GFP-Trap agarose. After the supernatant was discarded, GFP-Trap agarose was incubated with <i>E</i>. <i>coli</i>-expressed His-HA-NbSKP1.1, then the supernatant was discarded. GFP-Trap agarose was incubated with gradient dilutions (1, 1/2, 1/4) of His-βC1. Finally, agarose was washed and proteins were analyzed via SDS-PAGE and western blot assays using anti-GFP and anti-HA antibodies. Input was analyzed by the anti-His antibody (EASYBIO, China) and supernatant was analyzed by the anti-HA antibody. Intensity was detected through Total Lab TL120. (B) A confocal image of BiFC assays show that CLCuMuB βC1 interfered with the interaction between NbCUL1 and NbSKP1.1 <i>in vivo</i>. Photos were taken at 48 hpi. Bar scale represents 200 μm. (C) BiFC intensity (means±SEM, n = 4) was quantified by YFP fluorescence. Relative BiFC intensity was normalized to the control. The raw data were analyzed by two-sample <i>t</i>-test to show the significance level at 0.01 (**). (D) The protein level of cYFP-NbCUL1 and nYFP-NbSKP1.1 were checked with the polyclonal GFP antibody (Huaxin Bochuang, China). The PVDF membrane was stained with Ponceaux to visualize the large subunit of ribulose-1,5-bisphosphate as the loading control.</p

Silencing of <i>NbCUL1</i> enhances CLCuMuV DNA accumulation and results in typical disease symptoms.

<p>(A1 and A2) Six- to seven-week-old <i>N</i>. <i>benthamiana</i> plants were agroinoculated with CLCuMuV (CA) and βM2-<i>CUL1</i>F1 (A1), βM2-<i>CUL1</i>F2 (A2) or βM2-<i>βC1</i>F (as the control). (B1 and B2) Silencing of <i>NbCUL1</i> enhanced CLCuMuV DNA accumulation. Each group contained 7 plants. At 14 dpi, total DNA was extracted from each plant respectively and subjected to quantitative real-time PCR (means±SEM, n = 7) to quantify viral DNA accumulation. The internal reference method was used to calculate the relative amount of viral DNA. (C1 and C2) Real-time RT-PCR confirmed silencing of <i>NbCUL1</i>. Total RNA was extracted from each plant respectively and subjected to quantitative RT-PCR (means±SEM, n = 4) to quantify <i>NbCUL1</i> mRNA level. <i>Actin</i> was used as the internal reference. The raw data of (B1 and B2) and (C1 and C2) were analysed by two-sample <i>t</i>-test to show the significance level at 0.05 (*) and 0.01 (**). These experiments were repeated at least twice. (D1 and D2) 100% plants infected with CA+βM2-<i>CUL1</i>F1 (D1) or CA+βM2-<i>CUL1</i>F2 (D2) show severe symptoms at 21 dpi.</p