Journal: BMC Plant Biology

Article Title: Arabidopsis mutant sk156 reveals complex regulation of SPL15 in a miR156 -controlled gene network

doi: 10.1186/1471-2229-12-169

Figure Lengend Snippet: Summary of transgenic and mutant Arabidopsis lines characterized in this study

Article Snippet: 35S:SPL15n , Modified pBI121, 35S:SPL15n (miR156 sensitive), sk156 background , 4.7 fold less expresson for SPL15 . , Similar to sk156.

Techniques: Transgenic Assay, Mutagenesis, Construct, Gene Expression, Control, Expressing, Modification

Journal: Nature Communications

Article Title: A conserved oomycete effector RxLR23 triggers plant defense responses by targeting ERD15La to release NbNAC68

doi: 10.1038/s41467-024-50782-3

Figure Lengend Snippet: A Y2H confirmed that RxLR23 KM interacted with NbERD15La in yeast. Yeast transformants were first grown on SD/-Trp/-Leu (DDO), and then selected on SD/-Trp/-Leu/-His/-Ade/X-α-gal (QDO/X) for activating X-α-galactosidase activity. The images were photographed at 4 days after incubation. B Co-IP was used to examine the interaction of RxLR23 KM with NbERD15La in N. benthamiana leaves. Left panels confirm transient expression (+) RxLR23 KM -GFP and NbERD15La-FLAG. Equal protein loading is indicated by Ponceau staining (Ponceau S). Right panels show that RxLR23 KM -GFP is specifically combined with NbERD15La-FLAG. C RxLR23 KM uniquely interacts with NbERD15La upon Co-IP assay. Left panels confirmed transient expression (+) RxLR23 KM -GFP and NbERD15La/b/c-FLAG. Equal protein loading was indicated by Ponceau staining (Ponceau S). Right panels showed that RxLR23 KM -GFP was specifically combined with NbERD15La-FLAG. D NbERD15La specifically interacts with RxLR23 KM but not with its two allelic variants by Y2H. The images were photographed at 4 days after incubation. E The interaction of NbERD15La-FLAG with RxLR23 KM -GFP and its two allelic variants was detected using Co-IP in N. benthamiana leaves, respectively. Left panels confirmed transient expression of (+) RxLR23 KM -GFP, two allelic variants, and NbERD15La-FLAG alone. Equal protein loading was indicated by Ponceau staining (Ponceau S). Right panels showed that NbERD15La-FLAG was specifically combined with RxLR23 KM -GFP. F BiFC showed RxLR23 KM specifically interacted with NbERD15La in the nucleus and cytoplasm. Different pairs of constructs were co-expressed in N. benthamiana . The fluorescence was observed and imaged by confocal microscopy at 48 hpi. Scale bars, 20 μm. Each experiment was repeated at least three times. Source data are provided as a Source Data file.

Article Snippet: To generate pBinGFP2:RxLR23 KM , pBinGFP2:RxLR23 RM , pBinGFP2:RxLR23 RR , pBinGFP2:RxLR23 K93R , pBinGFP2:RxLR23 M320R , pBinGFP2:RxLR23 K93RM320R , pBinGFP2: NLS RxLR23, pBinGFP2: NES RxLR23, and pBinGFP2:INF1 (Supplementary Table ), the PCR product of them was purified and inserted into pBinGFP2 alone. pGADT7:CaERD15La/b/c, pGADT7:CaPABP, pCHF3301-3XFLAG:CaPABP, and pCHF3301-3XFLAG:CaERD15La/b/c (Supplementary Table ) were generated by cloning PCR product from C. annuum cDNA into pGADT7 and pCHF3301-3XFLAG alone using T4 DNA ligase (ACCURATE BIOTECHNOLOGY(HUNAN) CO., LTD, ChangSha, China). pBinGFP2:NbERD15La/b/c and pBinGFP2:NbNAC68a/b/c, pGADT7:NbERD15La/b/c, pCHF3301-3XFLAG:NbERD15La, pCHF3301-3XFLAG:NbNAC68a/b/c, pSPYCE:NbERD15La, pBI121-mCherry:NbNAC68a/b/c, pCAIMBIA1300-nLuc:NbERD15La, pCAIMBIA1300-cLuc:NbNAC68a/b/c, pGreenII62-SK:NbERD15La, pGreenII 62-SK:NbNAC68a/b/c, pGreenII 0800-Luc: PR1 , and pGreenII 0800-Luc: PR2 (Supplementary Table ) were generated by cloning PCR product from N. benthamiana cDNA into pBinGFP2, pGADT7, pCHF3301-3XFLAG, pSPYCE, pBI121-mCherry, pCAIMBIA1300-nLuc, pCAIMBIA1300-cLuc, pGreenII62-SK, and pGreenII 0800-Luc, respectively. pGBKT7:RxLR23 KM , pGBKT7:RxLR23 RM , pGBKT7:RxLR23 RR , pGBKT7: NLS RxLR23, pGBKT7: NES RxLR23, pSPYNE:RxLR23 KM , pCHF3301-3XFLAG:RxLR23 KM , and pCAIMBIA1300-nLuc:RxLR23 KM (Supplementary Table ) were generated by cloning PCR product from P. capsici into pGBKT7, pSPYNE, pCHF3301-3XFLAG, and pCAIMBIA1300-nLuc, respectively.

Techniques: Activity Assay, Incubation, Co-Immunoprecipitation Assay, Expressing, Staining, Construct, Fluorescence, Confocal Microscopy

Journal: Nature Communications

Article Title: A conserved oomycete effector RxLR23 triggers plant defense responses by targeting ERD15La to release NbNAC68

doi: 10.1038/s41467-024-50782-3

Figure Lengend Snippet: pBinGFP2:RxLR23 KM , pBinGFP2:NbERD15La, NbERD15La + RxLR23 KM , and pBinGFP2 were transiently expressed in S (TRV:NbERD15La) and NS (TRV:GFP) N. benthamiana leaves alone. The leaves were inoculated with P. capsici zoospores after expression of all these constructs at 24 h. A Typical lesion images of leaves were photographed under UV irradiation at 48 hpi ( n = 30 samples). B H 2 O 2 accumulation in N. benthamiana leaves was measured by DAB staining at 36 h ( n = 30 samples). C Mean diameter of lesions was measured at 48 hpi. Values are presented as mean ± SD, n = 30 samples. D Relative biomass of P. capsici was measured by qPCR at 48 hpi. Pcβ-actin and NbEF1α were identified as the most suitable reference genes for normalization. The ratio in the leaves inoculated with GFP was assigned to value of 1.0. Values are presented as mean ± SD, n = 3 independent experiments. E Relative levels of DAB staining were examined at 36 h. The relative staining of GFP was assigned to value 1.0. Values are presented as mean ± SD, n = 3 independent experiments. F Expected proteins size was detected by western blots using GFP-antibodies alone. Protein loading is indicated by Ponceau staining (Ponceau S). G Expression levels of both PR1 / 2 were detected by qRT-PCR at 48 hpi. NbEF1α of N. benthamiana was used as constitutively expressed endogenous control. Values are presented as mean ± SD, n = 3 independent experiments. H , I The variations of SA or ABA content were examined by HPLC-MS in N. benthamiana leaves after expressing each of these constructs at 24 h, respectively. Values are presented as mean ± SD, n = 3 independent experiments. The data in ( C – E , G – I ) were analyzed by ANOVA one-way comparison followed by least significant difference (LSD) test and different letters above the bars indicate a significant difference at p < 0.05. These experiments ( A – C , E , F ) were repeated three times with similar results. Source data are provided as a Source Data file.

Article Snippet: To generate pBinGFP2:RxLR23 KM , pBinGFP2:RxLR23 RM , pBinGFP2:RxLR23 RR , pBinGFP2:RxLR23 K93R , pBinGFP2:RxLR23 M320R , pBinGFP2:RxLR23 K93RM320R , pBinGFP2: NLS RxLR23, pBinGFP2: NES RxLR23, and pBinGFP2:INF1 (Supplementary Table ), the PCR product of them was purified and inserted into pBinGFP2 alone. pGADT7:CaERD15La/b/c, pGADT7:CaPABP, pCHF3301-3XFLAG:CaPABP, and pCHF3301-3XFLAG:CaERD15La/b/c (Supplementary Table ) were generated by cloning PCR product from C. annuum cDNA into pGADT7 and pCHF3301-3XFLAG alone using T4 DNA ligase (ACCURATE BIOTECHNOLOGY(HUNAN) CO., LTD, ChangSha, China). pBinGFP2:NbERD15La/b/c and pBinGFP2:NbNAC68a/b/c, pGADT7:NbERD15La/b/c, pCHF3301-3XFLAG:NbERD15La, pCHF3301-3XFLAG:NbNAC68a/b/c, pSPYCE:NbERD15La, pBI121-mCherry:NbNAC68a/b/c, pCAIMBIA1300-nLuc:NbERD15La, pCAIMBIA1300-cLuc:NbNAC68a/b/c, pGreenII62-SK:NbERD15La, pGreenII 62-SK:NbNAC68a/b/c, pGreenII 0800-Luc: PR1 , and pGreenII 0800-Luc: PR2 (Supplementary Table ) were generated by cloning PCR product from N. benthamiana cDNA into pBinGFP2, pGADT7, pCHF3301-3XFLAG, pSPYCE, pBI121-mCherry, pCAIMBIA1300-nLuc, pCAIMBIA1300-cLuc, pGreenII62-SK, and pGreenII 0800-Luc, respectively. pGBKT7:RxLR23 KM , pGBKT7:RxLR23 RM , pGBKT7:RxLR23 RR , pGBKT7: NLS RxLR23, pGBKT7: NES RxLR23, pSPYNE:RxLR23 KM , pCHF3301-3XFLAG:RxLR23 KM , and pCAIMBIA1300-nLuc:RxLR23 KM (Supplementary Table ) were generated by cloning PCR product from P. capsici into pGBKT7, pSPYNE, pCHF3301-3XFLAG, and pCAIMBIA1300-nLuc, respectively.

Techniques: Expressing, Construct, Irradiation, Staining, Western Blot, Quantitative RT-PCR, Control, Comparison

Journal: Nature Communications

Article Title: A conserved oomycete effector RxLR23 triggers plant defense responses by targeting ERD15La to release NbNAC68

doi: 10.1038/s41467-024-50782-3

Figure Lengend Snippet: The activity of NbERD15La was affected by SA or PAC. A Transcript levels of NbERD15La were measured by qRT-PCR after treatment with SA or PAC. Values are presented as mean ± SD, n = 3 independent experiments. B Expression of NbERD15La was detected using quantitative western blots in leaves following treatment with SA or PAC. Quantification of NbERD15La expression was calculated as relative intensity of NbERD15La bands. Protein loading is indicated by Ponceau S. Values are presented as mean ± SD, n = 3 independent experiments. C Neither SA nor PAC affected the interaction of RxLR23 KM with NbERD15La. Right panels showed expression (+) of NbERD15La-GFP, RxLR23 KM -FLAG, and GFP. Left panels showed the combination of RxLR23 KM and NbERD15La. The ratio of the band intensity of IP to input was shown in the bottom of IP panel. Equal protein loading was indicated by Ponceau S. D – F N. benthamiana treatment with single or sequential combinations of RxLR23 KM and SA in TRV:NbERD15La (S) or TRV:GFP (NS) plants. NS or S plants without any treatment were used as control. D Representative lesions were taken under UV irradiation at 48 h after P. capsici infection. Mean diameter of lesions was measured at 48 hpi. Values are presented as mean ± SD, n = 30 samples. E qPCR was used to measure the relative biomass on the ratios of P. capsici to S and NS leaves DNA at 48 hpi. The ratio of NS leaves without any treatment was assigned to value 1.0. Values are presented as mean ± SD, n = 3 independent experiments. F Expression levels of PR1 / 2 was detected by qRT-PCR at 48 hpi. Values are presented as mean ± SD, n = 3 independent experiments. The data in ( B , D – F ) were analyzed by ANOVA one-way comparison followed by least significant difference (LSD) test and different letters above the bars indicate a significant difference at p < 0.05. Pcβ-actin and NbEF1α were identified as the most suitable reference genes for normalization in P. capsici and N. benthamiana , respectively. These experiments ( B – D ) were repeated three times. Source data are provided as a Source Data file.

Article Snippet: To generate pBinGFP2:RxLR23 KM , pBinGFP2:RxLR23 RM , pBinGFP2:RxLR23 RR , pBinGFP2:RxLR23 K93R , pBinGFP2:RxLR23 M320R , pBinGFP2:RxLR23 K93RM320R , pBinGFP2: NLS RxLR23, pBinGFP2: NES RxLR23, and pBinGFP2:INF1 (Supplementary Table ), the PCR product of them was purified and inserted into pBinGFP2 alone. pGADT7:CaERD15La/b/c, pGADT7:CaPABP, pCHF3301-3XFLAG:CaPABP, and pCHF3301-3XFLAG:CaERD15La/b/c (Supplementary Table ) were generated by cloning PCR product from C. annuum cDNA into pGADT7 and pCHF3301-3XFLAG alone using T4 DNA ligase (ACCURATE BIOTECHNOLOGY(HUNAN) CO., LTD, ChangSha, China). pBinGFP2:NbERD15La/b/c and pBinGFP2:NbNAC68a/b/c, pGADT7:NbERD15La/b/c, pCHF3301-3XFLAG:NbERD15La, pCHF3301-3XFLAG:NbNAC68a/b/c, pSPYCE:NbERD15La, pBI121-mCherry:NbNAC68a/b/c, pCAIMBIA1300-nLuc:NbERD15La, pCAIMBIA1300-cLuc:NbNAC68a/b/c, pGreenII62-SK:NbERD15La, pGreenII 62-SK:NbNAC68a/b/c, pGreenII 0800-Luc: PR1 , and pGreenII 0800-Luc: PR2 (Supplementary Table ) were generated by cloning PCR product from N. benthamiana cDNA into pBinGFP2, pGADT7, pCHF3301-3XFLAG, pSPYCE, pBI121-mCherry, pCAIMBIA1300-nLuc, pCAIMBIA1300-cLuc, pGreenII62-SK, and pGreenII 0800-Luc, respectively. pGBKT7:RxLR23 KM , pGBKT7:RxLR23 RM , pGBKT7:RxLR23 RR , pGBKT7: NLS RxLR23, pGBKT7: NES RxLR23, pSPYNE:RxLR23 KM , pCHF3301-3XFLAG:RxLR23 KM , and pCAIMBIA1300-nLuc:RxLR23 KM (Supplementary Table ) were generated by cloning PCR product from P. capsici into pGBKT7, pSPYNE, pCHF3301-3XFLAG, and pCAIMBIA1300-nLuc, respectively.

Techniques: Activity Assay, Quantitative RT-PCR, Expressing, Western Blot, Control, Irradiation, Infection, Comparison

Journal: Nature Communications

Article Title: A conserved oomycete effector RxLR23 triggers plant defense responses by targeting ERD15La to release NbNAC68

doi: 10.1038/s41467-024-50782-3

Figure Lengend Snippet: pBinGFP2:NbNAC68a/b/c, pBinGFP2:NbERD15La, and pBinGFP2 were transiently expressed in N. benthamiana leaves alone, while NbERD15La + NbNAC68a/b/c is transiently co-expressed in N. benthamiana leaves. S represents TRV:NbERD15La N. benthamiana (Silenced plants). NS represents TRV:GFP N. benthamiana (Non-silenced plants). All tested N. benthamiana leaves were inoculated with P. capsici zoospores after expression of the corresponding constructs at 24 h. A Typical lesion images of leaves were photographed under UV irradiation at 48 hpi. Mean diameter of lesions was calculated at 48 hpi. Values are presented as mean ± SD, n = 30 samples. B Relative biomass of P. capsici was measured by qPCR at 48 hpi. Pcβ-actin and NbEF1α were identified as the most suitable reference genes for normalization. The ratio in NS leaves inoculated with GFP was assigned to value of 1.0. Values are presented as mean ± SD, n = 3 independent experiments. C Expression levels of PR1/2 were detected by qRT-PCR at 48 hpi. NbEF1α of N. benthamiana was used as constitutively expressed endogenous control. Values are presented as mean ± SD, n = 3 independent experiments. D , E The variations of SA or ABA content were examined by HPLC-MS in N. benthamiana leaves after above treatments at 24 hpi. Values are presented as mean ± SD, n = 3 independent experiments. F Expected protein sizes were detected by western blots using GFP-antibodies alone. Protein loading is indicated by Ponceau staining (Ponceau S). The data in Fig. A–E were analyzed by ANOVA one-way comparison followed by least significant difference (LSD) test and different letters above the bars indicate a significant difference at p < 0.05. These experiments ( A , F ) were repeated three times with similar results. Source data are provided as a Source Data file.

Article Snippet: To generate pBinGFP2:RxLR23 KM , pBinGFP2:RxLR23 RM , pBinGFP2:RxLR23 RR , pBinGFP2:RxLR23 K93R , pBinGFP2:RxLR23 M320R , pBinGFP2:RxLR23 K93RM320R , pBinGFP2: NLS RxLR23, pBinGFP2: NES RxLR23, and pBinGFP2:INF1 (Supplementary Table ), the PCR product of them was purified and inserted into pBinGFP2 alone. pGADT7:CaERD15La/b/c, pGADT7:CaPABP, pCHF3301-3XFLAG:CaPABP, and pCHF3301-3XFLAG:CaERD15La/b/c (Supplementary Table ) were generated by cloning PCR product from C. annuum cDNA into pGADT7 and pCHF3301-3XFLAG alone using T4 DNA ligase (ACCURATE BIOTECHNOLOGY(HUNAN) CO., LTD, ChangSha, China). pBinGFP2:NbERD15La/b/c and pBinGFP2:NbNAC68a/b/c, pGADT7:NbERD15La/b/c, pCHF3301-3XFLAG:NbERD15La, pCHF3301-3XFLAG:NbNAC68a/b/c, pSPYCE:NbERD15La, pBI121-mCherry:NbNAC68a/b/c, pCAIMBIA1300-nLuc:NbERD15La, pCAIMBIA1300-cLuc:NbNAC68a/b/c, pGreenII62-SK:NbERD15La, pGreenII 62-SK:NbNAC68a/b/c, pGreenII 0800-Luc: PR1 , and pGreenII 0800-Luc: PR2 (Supplementary Table ) were generated by cloning PCR product from N. benthamiana cDNA into pBinGFP2, pGADT7, pCHF3301-3XFLAG, pSPYCE, pBI121-mCherry, pCAIMBIA1300-nLuc, pCAIMBIA1300-cLuc, pGreenII62-SK, and pGreenII 0800-Luc, respectively. pGBKT7:RxLR23 KM , pGBKT7:RxLR23 RM , pGBKT7:RxLR23 RR , pGBKT7: NLS RxLR23, pGBKT7: NES RxLR23, pSPYNE:RxLR23 KM , pCHF3301-3XFLAG:RxLR23 KM , and pCAIMBIA1300-nLuc:RxLR23 KM (Supplementary Table ) were generated by cloning PCR product from P. capsici into pGBKT7, pSPYNE, pCHF3301-3XFLAG, and pCAIMBIA1300-nLuc, respectively.

Techniques: Expressing, Construct, Irradiation, Quantitative RT-PCR, Control, Western Blot, Staining, Comparison

Journal: Nature Communications

Article Title: A conserved oomycete effector RxLR23 triggers plant defense responses by targeting ERD15La to release NbNAC68

doi: 10.1038/s41467-024-50782-3

Figure Lengend Snippet: A , B pBinGFP2:RxLR23 KM , pBinGFP2:NbERD15La, pBinGFP2:NbNAC68a/b/c, pBinGFP2:NbERD15La + pBinGFP2:NbNAC68a/b/c, pBinGFP2:RxLR23 KM + pBinGFP2:NbERD15La, pBinGFP2:RxLR23 KM + pBinGFP2:NbNAC68a/b/c, pBinGFP2:RxLR23 KM + pBinGFP2:NbERD15La + pBinGFP2:NbNAC68a/b/c, or pBinGFP2:GFP was transiently expressed in N. benthamiana leaves and subsequently inoculated with P. capsici zoospores at 24 hpi. A Representative lesions were taken under UV irradiation at 48 hpi ( n = 30 samples). Mean diameter of lesions was measured at 48 hpi. Values are presented as mean ± SD. B Relative biomass of P. capsici was measured by qPCR at 48 hpi. Pcβ-actin and NbEF1α were identified as the most suitable reference genes for normalization. The ratio in the leaves inoculated with GFP was assigned to value of 1.0. Values are presented as mean ± SD, n = 3 independent experiments. C Expected protein sizes were detected by western blot. Protein loading is indicated by Ponceau S. D RxLR23 KM attenuates interaction of ERD15La and NbNAC68a/b/c. NbNAC68a/b/c-mCherry and NbERD15La-GFP were transiently co-expressed in N. benthamiana leaves along with increasing amounts of RxLR23 KM -FLAG or GST-FLAG. The ratio of NbNAC68a/b/c or RxLR23 KM immunoprecipitated by NbERD15La to their expression quantity was assigned to value 1.0 when NbNAC68a/b/c or NbERD15La with RxLR23 KM or GST were co-expressed into leaves in a 1:1:0.5 ratio. Equal protein loading is indicated by Ponceau S. E RxLR23 KM impairs NbNAC68a/b/c interaction with NbERD15La by LCI. MBP-nLUC and GST-cLUC were used as controls. RxLR23 KM and fusion proteins were co-expressed in leaves. Images were obtained at 2 days. LCI was evaluate interaction of NbERD15La with NbNAC68a/b/c in presence or absence of RxLR23 KM . Relative luminescence units (RLUs) were used to measure the luminous intensity. Values are presented as mean ± SD, n = 3 independent experiments. F RxLR23 KM weakens binding affinity of NbERD15La with NbNAC68a/b/c by MST. Values are presented as mean ± SD, n = 3 independent experiments. The data were analyzed by ANOVA one-way comparison followed by least significant difference (LSD) test. Different letters above the bars indicate a significant difference at p < 0.05. The experiments were repeated three times with similar results. Source data are provided as a Source Data file.

Article Snippet: To generate pBinGFP2:RxLR23 KM , pBinGFP2:RxLR23 RM , pBinGFP2:RxLR23 RR , pBinGFP2:RxLR23 K93R , pBinGFP2:RxLR23 M320R , pBinGFP2:RxLR23 K93RM320R , pBinGFP2: NLS RxLR23, pBinGFP2: NES RxLR23, and pBinGFP2:INF1 (Supplementary Table ), the PCR product of them was purified and inserted into pBinGFP2 alone. pGADT7:CaERD15La/b/c, pGADT7:CaPABP, pCHF3301-3XFLAG:CaPABP, and pCHF3301-3XFLAG:CaERD15La/b/c (Supplementary Table ) were generated by cloning PCR product from C. annuum cDNA into pGADT7 and pCHF3301-3XFLAG alone using T4 DNA ligase (ACCURATE BIOTECHNOLOGY(HUNAN) CO., LTD, ChangSha, China). pBinGFP2:NbERD15La/b/c and pBinGFP2:NbNAC68a/b/c, pGADT7:NbERD15La/b/c, pCHF3301-3XFLAG:NbERD15La, pCHF3301-3XFLAG:NbNAC68a/b/c, pSPYCE:NbERD15La, pBI121-mCherry:NbNAC68a/b/c, pCAIMBIA1300-nLuc:NbERD15La, pCAIMBIA1300-cLuc:NbNAC68a/b/c, pGreenII62-SK:NbERD15La, pGreenII 62-SK:NbNAC68a/b/c, pGreenII 0800-Luc: PR1 , and pGreenII 0800-Luc: PR2 (Supplementary Table ) were generated by cloning PCR product from N. benthamiana cDNA into pBinGFP2, pGADT7, pCHF3301-3XFLAG, pSPYCE, pBI121-mCherry, pCAIMBIA1300-nLuc, pCAIMBIA1300-cLuc, pGreenII62-SK, and pGreenII 0800-Luc, respectively. pGBKT7:RxLR23 KM , pGBKT7:RxLR23 RM , pGBKT7:RxLR23 RR , pGBKT7: NLS RxLR23, pGBKT7: NES RxLR23, pSPYNE:RxLR23 KM , pCHF3301-3XFLAG:RxLR23 KM , and pCAIMBIA1300-nLuc:RxLR23 KM (Supplementary Table ) were generated by cloning PCR product from P. capsici into pGBKT7, pSPYNE, pCHF3301-3XFLAG, and pCAIMBIA1300-nLuc, respectively.

Techniques: Irradiation, Western Blot, Immunoprecipitation, Expressing, Binding Assay, Comparison

Journal: Nature Communications

Article Title: A conserved oomycete effector RxLR23 triggers plant defense responses by targeting ERD15La to release NbNAC68

doi: 10.1038/s41467-024-50782-3

Figure Lengend Snippet: Working model illustrating how RxLR23 manipulates the NbERD15La-NbNAC68 subcomplex to activate plant defense response. A In the absence of RxLR23, the activity of NbNAC68 is restricted by binding with NbERD15La following suppresses ROS and SA production and enhances ABA accumulation, leading to blocked plant defense response. B In the presence of RxLR23, NbNAC68 is released from NbERD15La-NbNAC68 complex, which increases SA and ROS accumulation, and reduces ABA production, resulting in enhanced plant defense response and cell death.

Article Snippet: To generate pBinGFP2:RxLR23 KM , pBinGFP2:RxLR23 RM , pBinGFP2:RxLR23 RR , pBinGFP2:RxLR23 K93R , pBinGFP2:RxLR23 M320R , pBinGFP2:RxLR23 K93RM320R , pBinGFP2: NLS RxLR23, pBinGFP2: NES RxLR23, and pBinGFP2:INF1 (Supplementary Table ), the PCR product of them was purified and inserted into pBinGFP2 alone. pGADT7:CaERD15La/b/c, pGADT7:CaPABP, pCHF3301-3XFLAG:CaPABP, and pCHF3301-3XFLAG:CaERD15La/b/c (Supplementary Table ) were generated by cloning PCR product from C. annuum cDNA into pGADT7 and pCHF3301-3XFLAG alone using T4 DNA ligase (ACCURATE BIOTECHNOLOGY(HUNAN) CO., LTD, ChangSha, China). pBinGFP2:NbERD15La/b/c and pBinGFP2:NbNAC68a/b/c, pGADT7:NbERD15La/b/c, pCHF3301-3XFLAG:NbERD15La, pCHF3301-3XFLAG:NbNAC68a/b/c, pSPYCE:NbERD15La, pBI121-mCherry:NbNAC68a/b/c, pCAIMBIA1300-nLuc:NbERD15La, pCAIMBIA1300-cLuc:NbNAC68a/b/c, pGreenII62-SK:NbERD15La, pGreenII 62-SK:NbNAC68a/b/c, pGreenII 0800-Luc: PR1 , and pGreenII 0800-Luc: PR2 (Supplementary Table ) were generated by cloning PCR product from N. benthamiana cDNA into pBinGFP2, pGADT7, pCHF3301-3XFLAG, pSPYCE, pBI121-mCherry, pCAIMBIA1300-nLuc, pCAIMBIA1300-cLuc, pGreenII62-SK, and pGreenII 0800-Luc, respectively. pGBKT7:RxLR23 KM , pGBKT7:RxLR23 RM , pGBKT7:RxLR23 RR , pGBKT7: NLS RxLR23, pGBKT7: NES RxLR23, pSPYNE:RxLR23 KM , pCHF3301-3XFLAG:RxLR23 KM , and pCAIMBIA1300-nLuc:RxLR23 KM (Supplementary Table ) were generated by cloning PCR product from P. capsici into pGBKT7, pSPYNE, pCHF3301-3XFLAG, and pCAIMBIA1300-nLuc, respectively.

Techniques: Activity Assay, Binding Assay

Journal: Frontiers in Plant Science

Article Title: Multiomics studies with co-transformation reveal microRNAs via miRNA-TF-mRNA network participating in wood formation in Hevea brasiliensis

doi: 10.3389/fpls.2023.1068796

Figure Lengend Snippet: Validation of the targeting of HbrCAD1 by its predicted miRNA novel_28 in Nicotiana benthamiana . β-actin was selected as internal reference; data are means ± standard error of three independent biological replicates. (A) Alignment of hbr-miR482c and HbrCAD1 . (B) The plasmids pBI121-miR482c and GH714-013930-pBI121 used for the assay. (C) Principle of transient infiltration of N. benthamiana leaves with an Agrobacterium cell suspension. (D) Relative HbrCAD1 expression levels in different samples. Data are shown as Log2 (fold-change), with the expression of HbrCAD1 from the pair pBI121 + 35S:HbrCAD1 set to 1.

Article Snippet: The resulting PCR product was digested with BamH I and Sac I and ligated into the vector pBI121 downstream of the cauliflower mosaic virus (CaMV) 35S promoter (Shanghai Generay Biotech; ).

Techniques: Suspension, Expressing