Additional file 1 of The Fungal Effector Avr-Pita Suppresses Innate Immunity by Increasing COX Activity in Rice Mitochondria
Creators: name: Han, Jingluan
name: Wang, Xiaoyu
name: Wang, Fengpin
name: Zhao, Zhe
name: Li, Gousi
name: Zhu, Xiaoyuan
name: Su, Jing
name: Chen, Letian
Publisher: name: figshare
Year: 2021
Subjects: subject: Plant Biology
subject: FOS: Biological sciences

description: Additional file 1: Figure S1 Ectopic expression of Avr-Pita in rice. (a) Diagram of the PXVE::Avr-Pita and PUbi::Avr-Pita constructs. In these constructs, a truncated Avr-Pita145–672 fragment encoding mature protease is driven by the estradiol-inducible promoter XVE or the maize Ubiquitin (Ubi) promoter. SP: signal peptide; Pro: predicted prosequence. (b) The growth and developmental morphology of PUbi::Avr-Pita plants were not obviously affected in these lines. Scale bars: 10 cm. (c) Transcript levels of Avr-Pita was measured by RT-PCR in PXVE::Avr-Pita transgenic suspension cell lines after estradiol treatment. (d) Transcript levels of Avr-Pita was measured by RT-PCR in PUbi::Avr-Pita transgenic lines. (e) The expression of Avr-Pita in PXVE::Avr-Pita transgenic suspension cell lines after estradiol treatment using qRT-PCR. (f) The expression of defense-response gene OsPAL1 in PXVE::Avr-Pita suspension cell lines after estradiol treatment using qRT-PCR. (g) Overexpression of Avr-Pita in PUbi::Avr-Pita transgenic lines using qRT-PCR. OsACTIN served as a control to normalize the expression levels of target genes. Data are shown as mean ± SD (**P &lt; 0.01, n = 3). Figure S2 Avr-Pita specifically binds to the conserved domains of OsCOX11. (a) Amino acid sequence alignment of COX11 orthologs OsCOX11 (O. sativa, XP_006650503.1), AtCOX11 (A. thaliana, AAG00893), ScCOX11 (S. cerevisiae, NP_015193), and MoCOX11 (M. oryzae, XP_003717808). Six β sheets (indicated by a single underline and numbered 1–6) and Cu-binding core region CFCF (indicated by four triangles ▲) are present in the conserved region of COX11. Residues 140–220 of OsCOX11, which are responsible for the interaction with Avr-Pita, are double underlined. The critical region OsCOX11199–220 is labelled with boxes. (b) Avr-Pita specifically interacts with rice OsCOX11 in a Y2H assay. Yeast cells were cultured on selective medium SD-LW or SD-AHLW; cell growth on SD-AHLW indicates a positive interaction. Figure S3 Avr-Pita and OsCOX11 co-localize to the mitochondria in onion epidermal cells. (a) Avr-Pita:YFP was introduced into onion epidermal cells by particle bombardment and stained with the mitochondrial dye MitoTracker. (b) Avr-Pita:YFP and OsCOX11:mCherry were transiently introduced into onion epidermal cells by particle bombardment. Scale bar = 50 μm. Figure S4 Characterization of OsCOX11 transgenic plants and pathogen resistance of OsCOX11-RNAi plants. (a) Expression levels of OsCOX11 in PUbi::OsCOX11 lines; OsACTIN served as an internal control. Data are shown as mean ± SD (**P &lt; 0.01, n = 3). (b) Genotypes of heterozygous oscox11/OsCOX11 lines carrying a “T” or “C” base insertion. (c) Expression levels of OsCOX11 in oscox11/OsCOX11 plants. OsACTIN served as an internal control. Data are shown as mean ± SD (**P &lt; 0.01, n = 3). (d) Expression levels of OsCOX11 in OsCOX11-RNAi plants. OsACTIN served as an internal control. Data are shown as mean ± SD (**P &lt; 0.01, n = 3). (e) Disease symptoms of OsCOX11-RNAi transgenic plants at 12 dpi inoculated with M. oryzae isolate 13–219. (f) Lesion area in OsCOX11-RNAi transgenic plants at 12 dpi inoculated with M. oryzae isolate 13–219. Data are shown as mean ± SD (**P &lt; 0.01, n &gt; 12). (g) Relative fungal biomass on inoculated leaves at 12 dpi, as determined by qPCR. Data are shown as mean ± SD (**P &lt; 0.01, n = 3). Figure S5 OsCOX11 expression in response to chitin and M. oryzae treatment. (a) The expression levels of OsCOX11 in PUbi::Avr-Pita and WT plants after chitin treatment. (b) The expression levels of OsCOX11 in rice seedlings in response to M. oryzae compatible strain 08-T13 inoculation at the indicated time points. OsACTIN served as an internal control. Data are shown as mean ± SD (**P &lt; 0.01, n = 3). Table S1 CCandidates of Avr-Pita interacting protein screened by Y2H. Table S2 Primers used in this study.