The phytopathogenic virulent effector protein RipI induces apoptosis in budding yeast Saccharomyces cerevisiae

Accepted Manuscript The phytopathogenic virulent effector protein RipI induces apoptosis in budding yeast Saccharomyces cerevisiae Meng-ying Deng, Yun-hao Sun, Pai Li, Bei Fu, Dong Shen, Yong-jun Lu PII:

S0041-0101(16)30267-7

DOI:

10.1016/j.toxicon.2016.09.006

Reference:

TOXCON 5456

To appear in:

Toxicon

Received Date: 29 June 2016 Revised Date:

1 September 2016

Accepted Date: 6 September 2016

Please cite this article as: Deng, M.-y., Sun, Y.-h., Li, P., Fu, B., Shen, D., Lu, Y.-j., The phytopathogenic virulent effector protein RipI induces apoptosis in budding yeast Saccharomyces cerevisiae, Toxicon (2016), doi: 10.1016/j.toxicon.2016.09.006. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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The phytopathogenic virulent effector protein RipI induces

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apoptosis in budding yeast Saccharomyces cerevisiae

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Meng-ying Deng, Yun-hao Sun, Pai Li, Bei Fu, Dong Shen and Yong-jun Lu*

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School of Life Sciences and Biomedical Center, Sun Yat-sen University, No. 135 Xingang road

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west, Guangzhou 510275, China.

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* Corresponding author. School of Life Sciences and Biomedical Center, Sun Yat-sen

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University, No. 135 Xingang road west, Guangzhou 510275, China. Phone: +86-20-84110778. E-mail: [email protected] (Yong-jun Lu).

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ACCEPTED MANUSCRIPT Abstract

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Virulent protein toxins secreted by the bacterial pathogens can cause cytotoxicity by

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various molecular mechanisms to combat host cell defense. On the other hand, these

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proteins can also be used as probes to investigate the defense pathway of host innate

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immunity. Ralstonia solanacearum one of the most virulent bacterial phytopathogens,

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translocates more than 70 effector proteins via type III secretion system during

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infection. Here, we characterized the cytotoxicity of effector RipI in budding yeast

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Saccharomyce scerevisiae, an alternative host model. We found that over-expression

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of RipI resulted in severe growth defect and arginine (R) 117 within the predicted

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integrase motif was required for inhibition of yeast growth. The phenotype of death

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manifested the hallmarks of apoptosis. Our data also revealed that RipI-induced

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apoptosis was independent of Yca1 and mitochondria-mediated apoptotic pathways

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because ∆yca1 and ∆aif1 were both sensitive to RipI as compared with the wild type.

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We further demonstrated that RipI was localized in the yeast nucleus and the

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N-terminal 1-174aa was required for the localization. High-throughput RNA

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sequencing analysis showed that upon RipI over-expression, 101 unigenes of yeast

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ribosome presented lower expression level, and 42 GO classes related to the nucleus

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Abbreviations: FCM, flow cytometer; FITC, fluorescein isothiocyanate; NLS, nuclear localization signal; PI, propidium iodide; rip, Ralstonia injected protein; SC, synthetic complete; TEM, transmission electron microscopy; T3SS, type III secretion system; TUNEL, terminal-deoxynucleotidyl transferase mediated nick end labeling.

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ACCEPTED MANUSCRIPT or recombination were enriched with differential expression levels. Taken together,

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our data showed that a nuclear-targeting effector RipI triggers yeast apoptosis,

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potentially dependent on its integrase function. Our results also provided an

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alternative strategy to dissect the signaling pathway of cytotoxicity induced by the

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protein toxins.

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Key words:

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Apoptosis

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Effector protein

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Ralstonia solanacearum

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RipI

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RNA-seq

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Saccharomyces cerevisiae

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1. Introduction

The bacterium Ralstonia solanacearum can infect more than 200 species of

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plants worldwide (Genin and Boucher, 2004; Hayward, 1991), including several

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major economic crops and vegetables throughout the world (Genin and Boucher, 2004;

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Wicker et al., 2007). R. solanacearum is the most destructive bacterial phytopathogen

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and its infection causes lethal bacterial wilt disease in plants (Elphinstone, 2005;

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Mansfield et al., 2012). During infection, R. solanacearum secretes a number of

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effector proteins via type III secretion system (T3SS) to combat plant host defense

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system (Buttner D, 2002; Mukaihara et al., 2010; Zolobowska and Van Gijsegem,

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2006). For example, the effector proteins belonging to the Gala family with F-box and

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leucine-rich repeat domains have been reported to be required for complete virulence

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(Angot et al., 2006; Remigi et al., 2011). PopP1 is homologous to the members of the

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YopJ/AvrRxv family and acts as a typical avirulent protein (Muriel Lavie, 2002).

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PopP2 belongs to the YopJ-like family with acetyl transferase activity and interacts

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with RRS1-R in the nucleus of the plant cells (Bernoux et al., 2008; Deslandes et al.,

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2003; Tasset et al., 2010). Sole et al. reported that effectors belonging to the AWR

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family induce plant cell death and are essential for virulence (Sole et al., 2012).

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However, the functions of most of the effector proteins have not yet been evaluated.

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R. solanacearum GMI1000 strain is one of the most widely distributed strains.

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During infection, it delivers more than 70 effectors via T3SS to cause wilting in plants

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(Cunnac et al., 2004; Mukaihara et al., 2010; Peeters et al., 2013; Poueymiro and

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Genin, 2009). RipI is one of the effector proteins encoded by ripI (rsc0041), a rip 3

ACCEPTED MANUSCRIPT (Ralstonia injected protein) gene located on the chromosome (Mukaihara and Tamura,

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2009; Peeters et al., 2013). The translocation of RipI has been confirmed by a Cya

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reporter system (Mukaihara et al., 2010). However, the biochemical function of RipI

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remains completely unknown (Peeters et al., 2013).

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As a simple and sequenced eukaryotic organism with significant homology to

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plant and mammalian cells, the yeast Saccharomyces cerevisiae has been used as an

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ideal model system to study various intracellular mechanisms (Forsburg, 2005)

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including effector protein functions in the whole cell (Curak et al., 2009; Munkvold et

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al., 2008; Slagowski et al., 2008). It serves as an alternative in vivo system to validate

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or reveal biochemical functions of several effector proteins from various bacterial

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pathogens such as Yersinia enterocolitica (Lesser and Miller, 2001), Legionella

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pneumophila (Bennett et al., 2013; Heidtman et al., 2009; O'Brien et al., 2015),

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Magnaporthe oryzae (Cesari et al., 2013) and Vibrio cholera (Seward et al., 2015).

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The purpose of the present study was to investigate the cytotoxicity of

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over-expressed RipI on yeast cells. We provided evidence that RipI induces apoptotic

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cell death in yeast that is independent of Yca1p and Aif1p, two conserved yeast

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apoptosis pathways. Instead, DNA damage-related apoptosis based on the evidence of

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nuclear localization of RipI and high-throughput RNA sequencing analysis may be

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deduced.

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2. Materials and Methods

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2.1 Strains, media, growth conditions and standard techniques

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ACCEPTED MANUSCRIPT Escherichia. coli strain DH5α (F− (φ80dlac∆M15) (lacZYA-argF∆U169) endA1

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recA1 hsdR17 deoR thi-1 supE44 gyrA96 (Nalr) relA1) and R. solanacearum strain

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GMI1000 were used in this study. E. coli cells were grown aerobically in LB medium

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at 37°C. R. solanacearum cells were routinely cultivated in CPG medium at 30°C.

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Antibiotics were used as necessary: ampicillin, 100 mg/mL; chloramphenicol, 200

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mg/mL; kanamycin, 50 mg/mL. The wild type S. cerevisiae strain W303-1A (MATa

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ade2-1 ura3-1 his3-11 trp1-1 leu2-3 leu2-112 can1-100) was used in this study and as

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the parental strain for aif1 and yca1 deletion. The untransformed yeast cells were

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cultured in YPD medium at 30°C for the preparation of competent cells. The

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transformed yeast cells harboring the recombinant plasmids were grown at 30°C in

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synthetic complete (SC) media supplemented with glucose (SCD) or galactose (SCG)

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to a final concentration of 2%. Over-expression of the genes was induced by galactose.

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The molecular cloning was carried out according to standard protocols (Invitrogen).

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2.2 Plasmids construction and yeast transformation

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For expression of recombinant RipI and its mutants in yeast cells, commercial

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yeast expression plasmid pYES2/NTA and pYX223-RFP were used. The ripI gene,

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the ints fragment (a predicted integrase domain within RipI) and the mutant genes

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ripI N, ripI

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DNA as a template. The mutant genes ripI

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obtained by fusion PCR. The ripI gene, the ints fragment and all their mutants were

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cloned into the pYES2/NTA using EcoRI and XbaI sites to create pYES2-ripI,

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were PCR amplified using R. solanacearum strain GMI1000 genomic

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, ripIR117G, ripIF139G and intsR117G were

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pYES2-ripI C,

pYES2-ripIF139G, and pYES2-intsR117G, respectively. The mutations were verified by

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sequencing and Western-blot analysis. For subcellular co-localization experiments,

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the mitochondrial Pre fragment was excised from pYX223-RFP by EcoRI and BamHI

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restriction endonucleases. Subsequently, the ripI gene, the mutant genes ripI N, ripI C,

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ripI

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pYX223-ripI,

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pYX223-ripIR117G, respectively. Primer sequences were presented in Table S1.

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pYES2/NTA and pYX223-RFP -based expression vectors carrying RipI and its

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mutants were transformed into the competent cells of S. cerevisiae strains W303-1A,

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∆yca1 and ∆aif1, respectively, using the lithium acetate method (Gietz and Schiestl,

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2007). Then the cells were plated onto SCD-Ura solid media and incubated at 30 °C

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for 3 to 5 days for transformants growth.

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and ripIR117G were inserted into the EcoRI and BglII sites to create pYX223-ripI

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pYX223-ripI C,

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2.3 Yeast growth inhibition assay

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pYES2-ripIR117G,

pYES2-ints,

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pYES2-ripI

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Yeast cells containing respective plasmids were grown in SCD-Ura liquid

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medium at 30°C with agitation, to an OD600 nm of 1-1.3. After induction by

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galactose for 6 h, the cells were 10-fold serially diluted. 4 µL of each dilution was

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spotted onto SCD-Ura and SCG-Ura plates. The plates were incubated at 30°C and

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photographed at 120 h using Sony APS-C camera. The remaining cells were used for

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Western-blot analysis.

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2.4 DAPI staining and TEM observation Yeast cells containing respective plasmids were grown in SCG-Ura liquid

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medium at 30°C with agitation for 12 h followed by the treatment of 40 mM formic

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acid upon W303-1A: pYES2/NTA cells for 45 min. The cells were then harvested,

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washed twice with cold PBS buffer, fixed in 75% alcohol for at least 10 min,

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centrifuged and washed again with cold PBS buffer. DAPI was added to a final

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concentration of 2 µg/mL. After incubation for 30 min, the morphology of the cell

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nuclei was observed under a Nikon fluorescence microscope. For transmission

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electron microscopy (TEM) observation, the cells were harvested, washed twice with

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cold PBS buffer and then fixed with 25% glutaraldehyde. The subsequent sample

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preparation was same as above. The morphology of the cell was observed and

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photographed by JEM1400 electron microscope.

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2.5 Annexin V-FITC and PI staining

Exposed phosphatidylserine was detected by fluorescein isothiocyanate

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(FITC)-conjugated Annexin V using an Annexin V-FITC Apoptosis Detection Kit

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(Becton and Dickinson). Yeast cells containing respective plasmids were grown in

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SCG-Ura liquid medium at 30°C for 10 h with agitation followed by the treatment of

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40 mM formic acid upon W303-1A: pYES2/NTA cells for 45 min. Then, the cells

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were harvested, washed and resuspended in sorbitol buffer (1.2 M sorbitol, 0.5 mM

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MgCl2, 53 mM potassium phosphate, pH 6.8). The cell wall was digested with 20

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U/mL lyticase for 100 min at 37°C. Subsequently, the cells were harvested, washed

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ACCEPTED MANUSCRIPT and resuspended in 100 µL binding buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM

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CaCl2, 1.2 M sorbitol, pH 7.4). 5 µL Annexin V-FITC and 5 µL Propidium Iodide (PI)

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were added to 90 µL cell suspension and incubated for 30 min at room temperature in

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the dark. The cells were then harvested and washed for flow cytometer (FCM)

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analysis.

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2.6 TUNEL assay

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DNA strand breaks were monitored by terminal-deoxynucleotidyl transferase

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mediated nick end labeling (TUNEL) using a TUNEL Apoptosis Assay Kit (Roche).

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Yeast cells containing respective plasmids were grown in SCG-Ura liquid medium at

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30°C with agitation for 14 h. Then W303-1A: pYES2/NTA cells were treated with 40

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mM formic acid for 45 min. The cells were harvested, washed and resuspended in

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sorbitol buffer. The cell wall was digested with 20 U/mL lyticase followed by fixation

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with 75% ethanol for at least 10 min. Subsequently, the cells were blocked with 3%

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H2O2 in methanol for 10 min at 15-25°C and washed twice with cold PBS buffer.

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Then, the cells were permeabilized with 0.1% TritonX-100 and 0.1% sodium citrate

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for 2 min at 2-8°C and washed. Finally, the cells were incubated for 120 min at 37°C

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in a dark and humid environment with 50 µL TUNEL reaction mixture prepared

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according to the manufacturer’s protocol. The cells were harvested, resuspended in

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PBS, observed under a fluorescence microscope and used for FCM analysis.

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2.7 Subcellular co-localization in yeast cells

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ACCEPTED MANUSCRIPT Yeast cells containing respective plasmids were grown in SCG-His liquid

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medium with agitation at 30°C for 8 h. Then, the cells were harvested, washed twice

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with cold PBS buffer, fixed in 75% alcohol for 10 min, centrifuged and washed again.

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DAPI was added to a final concentration of 2 µg/mL and the cells were incubated for

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30 min, following which, the subcellular co-localization of the RipI protein and its

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mutants was monitored using a Nikon fluorescence microscope.

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2.8 High-throughput RNA sequencing

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Yeast cells containing respective plasmids were grown in SCD-Ura liquid

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medium at 30°C with agitation until OD600 nm of 1-1.3. After induction with

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galactose for 10 h, the cells were harvested, washed, and placed in liquid nitrogen for

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RNA extraction. Total RNA was extracted using TRIzol reagent according to the

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manufacturer’s instructions (Invitrogen). The integrity and purity of the RNA content

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were estimated by Nanodrop2000. The qualified RNA samples were used for cDNA

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synthesis. Poly (A) mRNA was isolated using oligodT beads. All mRNA were

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fragmented into 200 bp by fragmentation buffer. First-strand cDNA was generated

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using random hexamers and reverse transcriptase, then the second-strand was

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obtained. The cDNA fragments were repaired using the End Repair Mix and washed

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with EB buffer for end reparation poly (A) addition. PCR amplification was

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performed on suitable template fragments. Finally, the cDNA library of yeast was

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constructed and sequenced on the Illumina HiSeq 4000.

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3. Results and Discussion

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3.1 Over-expression of RipI leads to severe growth inhibition in yeast To determine the cytotoxicity of RipI, the gene was cloned into the vector

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pYES2/NTA, generating pYES2-ripI, wherein the transcription of the ripI gene is

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controlled by the inducible GAL promoter, which is repressed by glucose and induced

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by galactose (Giniger et al., 1985). pYES2-ripI was then transformed into the yeast S.

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cerevisiae strain W303-1A and over-expressed, respectively. As shown in Fig. 1, cells

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expressing ripI exhibited severe growth defects, indicating the high cytotoxicity of

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RipI to yeast cells.

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Fig. 1. Over-expression of RipI protein causes severe growth defects in yeast cells.

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Yeast cells harboring pYES2/NTA or pYES2-ripI were 10-fold serially diluted, and

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aliquots were spotted onto SCD-Ura and SCG-Ura plates, respectively. Images of the

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resultant plates were captured after 120 h incubation at 30°C.

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3.2 RipI-induced cell death exhibits typical apoptosis phenotypes in S. cerevisiae

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Since the ectopic expression of RipI led to growth inhibition, it was imperative to

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examine whether RipI regulated cell death. To address this possibility, we first

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observed the morphology of the cells with TEM. The W303-1A: pYES2-ripI cells 10

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condensed and marginalized chromatin compared with the W303-1A: pYES2/NTA

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cells (Fig. 2A-a). This indicated a typical apoptosis phenotype similar to that induced

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by formic acid (Fig. 2A-d) (Du et al., 2008).

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Moreover, DAPI staining showed an aberrant nuclear phenotype with rippled or

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creased edge and condensed chromatin in W303-1A: pYES2-ripI cells, similar to

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W303-1A: pYES2/NTA cells treated with formic acid, as compared with W303-1A:

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pYES2/NTA cells without formic acid (Fig. 2B).

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Fig. 2. RipI-caused cell death characteristics of apoptotic phenotypes in yeast cells. (A)

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TEM results show morphologically changed characteristics of apoptosis. (a)

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W303-1A: pYES2/NTA as a negative control, (b-c) yeast cells with RipI

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over-expression for 12 h, and (d) W303-1A: pYES2/NTA treated with 40 mM formic 11

ACCEPTED MANUSCRIPT acid for 45 min as a positive control. The arrow in (a) shows the integrity of nucleus.

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Arrows in (c) and (d) show nuclear chromatin condensation. (B) DAPI staining

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reveals an irregular morphology of nucleus in cells with RipI over-expression for 12 h

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(b), as well as in W303-1A: pYES2/NTA treated with 40 mM formic acid for 45 min

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(a), compared with W303-1A: pYES2/NTA without formic acid treatment (c).

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Yeast also possesses the hallmarks of apoptosis such as phosphatidylserine

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externalization, chromatin condensation and DNA fragmentation (Madeo et al., 2004).

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To further confirm the RipI-associated cell death manifests the hallmarks of apoptosis,

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we first quantified the phosphatidylserine externalization and determined the cellular

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membrane integrity by Annexin V-FITC and PI co-staining (van Engeland et al.,

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1998). As shown in Fig. 3, about 43.2±8.70% of W303-1A: pYES2-ripI cells were

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Annexin V-FITC positive, higher than 12.9±5.40% of W303-1A: pYES2/NTA cells.

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As a positive control, approximate 86.18±3.35% of W303-1A: pYES2/NTA cells

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treated with formic acid were stained by Annexin V-FITC.

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Fig. 3. Phosphatidylserine externalization analysis of RipI-induced cell death. (A)

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Flow cytometry results of Annexin V-FITC staining. Yeast cells were induced by

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galactose for 10 h, then treated with or without 40 mM formic acid for 45 min.

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Approximate 104 cells were analyzed in each sample. (B) Quantification of the

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Annexin V-FITC or PI positive cells. The data represents the mean ± SEM of three

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independent experiments (P<0.01). FA: formic acid.

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Next, we detected the DNA fragmentation in RipI-expressing cells by TUNEL

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assay (Cuello-Carrión and Ciocca, 1999). As shown in Fig. 4A, fluorescence

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microscopy results revealed that, compared with the control, the vast majority of

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ACCEPTED MANUSCRIPT RipI-expressing cells as well as W303-1A: pYES2/NTA cells treated with formic acid

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were TUNEL positive. These results were further confirmed by flow cytometry assay

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(Fig. 4B, C), in which 66.10±8.82% of W303-1A: pYES2-ripI cells were TUNEL

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positive, whereas only 18.39±10.55% of W303-1A: pYES2/NTA cells. These results

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further strongly indicated that over-expression of RipI induced apoptotic cell death in

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S. cerevisiae.

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(The figure 4 file is larger than 10 MB and is uploaded separately)

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Fig. 4. DNA fragmentation revealed by TUNEL assay. (A) Fluorescence micrographs

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show TUNEL (+) (green) and TUNEL (-) cells. Yeast cells harboring pYES2-ripI or

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pYES2/NTA were induced with galactose for 14 h and then treated or not with 40 mM

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formic acid for 45 min. (B) Flow cytometric analysis of DNA fragmentation by

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TUNEL assay. Approximate 104 cells were analyzed for each sample. (C)

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Quantification of the percentage of TUNEL positive cells. The data represents the

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mean ± SEM of three independent experiments (P<0.01). FA: formic acid.

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3.3 RipI-induced apoptotic cell death in S. cerevisiae is independent of Yca1p and

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Aif1p

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The progress of yeast apoptosis is mediated by many intrinsic proteins. Among

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them, the metacaspase homolog Yca1 (Yeast Caspase-1) and the apoptosis-inducing

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factor Aif1 (Apoptosis-inducing factor-1) are the key regulators (Candé et al., 2002;

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Madeo et al., 2002; Mazzoni and Falcone, 2008; Wissing et al., 2004). To investigate

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ACCEPTED MANUSCRIPT whether these two proteins were involved in RipI-induced cell death, two null deletion

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strains, ∆yca1 and ∆aif1, as well as wild type were transformed with pYES2-ripI or

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pYES2/NTA, respectively. Cytotoxicity of the different constructs in yeast was

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determined by yeast growth inhibition assay. As shown in Fig. 5, over-expression of

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RipI in both of ∆yca1 and ∆aif1 did not elevate the survival rate of yeast cells as

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compared with the wild type. Because Yca1 is a key constituent in hydrogen

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peroxide-mediated apoptosis pathway (Madeo et al., 2002; Uren et al., 2000), while

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Aif1 mediates mitochondria apoptosis pathway under different stress conditions

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(Candé et al., 2002; Zamzami et al., 1995; Zamzami et al., 1996), these results

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indicated that RipI-induced apoptosis is independent of both Yca1 and Aif1 mediated

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pathways.

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Fig. 5. Growth inhibition assay on wild type and mutant yeast strains upon RipI

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over-expression. The transformants were 10-fold serially diluted and spotted onto

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SCD-Ura and SCG-Ura plates, respectively. Images of the resultant plates were

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captured after 120 h incubation at 30°C.

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3.4 Analysis of sequence in RipI that is required for growth inhibition in yeast To determine whether RipI activity relied on the putative domains, the sequence

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of RipI was analyzed by PHYRE2 (Protein Homology/ analogY Recognition Engine

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V 2.0). An ints integrase-like domain from N52 to N174 at the N-terminus of the

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protein with high confidence was identified (Fig. 6A). It has been known that ints

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integrase functions in controlling and regulating the site-specific recombination of

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KplE1 prophage (Panis et al., 2010a; Panis et al., 2010b). The alignment of ints

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integrase-like fragments among five bacterial species by ClustalX2 indicated that the

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R117 and F139 residues are the two conserved amino acids (Fig. 6B), suggesting that

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these may play a potential role in RipI functionality.

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To determine whether the ints integrase-like fragment contributes to RipI activity,

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deletion of the fragment and point mutation of the two conserved amino acids were

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constructed. As showed in Fig. 6C, the RipI with truncation of the ints fragment was

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not lethal to the yeast cells when compared with the control, while the expression of

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ints fragment alone showed high toxicity (+++), suggesting its pivotal role in RipI

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activity. Furthermore, R117G, but not F139G mutation, both in the full length and ints

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fragment rescued the growth inhibition, demonstrating that R117 residue is necessary

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for the function of ints fragment. On the contrary, RipI with N-terminal 1-51aa

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deletion efficiently inhibited the growth of yeast cells (Fig. 6C, second row), similar

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to the full-length protein (Fig. 1), indicating a minor role of this fragment. Deletion

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and mutated fragments were expressed efficiently in yeast cells as confirmed by

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Western-blot (Fig. 6D).

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Fig. 6. Identification of sequences in RipI required for inhibiting yeast growth. (A)

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Predicted structure of R. solanacearum effector RipI protein. Amino acid residue

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numbers are showed. Blue-shaded boxes indicate the ints integrase-like fragment from

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N52 to N174. N and C indicate the N-terminus and C-terminus of the protein. (B)

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ACCEPTED MANUSCRIPT Alignment of the protein sequence of ints integrase-like fragments among five

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different bacterial species, including E.coli CPS-53 (a), Klebsiella pneumoniae SA1

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(b). Bradyrhizobium BTAi1 (c), Mesorhizobium australicum WSM2073 (d), and R.

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solanacearum RipI (e). The arginine (R) on 117 (red box) and the phenylalanine (F)

348

on 139 (green box) are highly conserved across evolution. (C) Fresh cultures of wild

349

type strain W303-1A transformed with pYES2/NTA or with the pYES2-ripI

350

pYES2-ripI

351

pYES2-rip C, were 10-fold serially diluted and spotted onto SCD-Ura and SCG-Ura

352

plates, respectively. Images of the resultant plates were captured after 120 h

353

incubation at 30°C. (D) Identification of the mutated RipI proteins by Western-blot

354

analysis, using monoclonal anti-Xpress antibodies to detect the Express tag (1:5000,

355

Life Technologies). After reaction with secondary antibody goat-anti-mouse (1:2000)

356

for 2 h, the protein bands were visualized by FluorChem Q according to the

357

manufacturer’s protocol.

,

, pYES2-ripIR117G, pYES2-ripIF139G, pYES2-ints, pYES2-intsR117G and

M AN U

TE D

EP

3.5 Subcellular co-localization of RipI and its mutants in yeast cells

AC C

360

N

SC

ints

358 359

RI PT

344

To further investigate the underlying mechanism of RipI-induced apoptotic cell

361

death, we checked the subellular localization of RipI and its mutants in yeast cells. To

362

this end, ripI and its mutants ripI N, ripI

363

vector pYX223-RFP. The resulting plasmids pYX223-ripI, pYX223-ripI N,

364

pYX223-ripI

365

W303-1A, respectively. After the expression of ripI and its mutants, the localization

ints

, ripIR117G and ripI

ints

, pYX223-ripIR117G and pYX223-ripI

18

C

C

were cloned into the

were transformed into

ACCEPTED MANUSCRIPT 366

of the corresponding proteins was assessed by fluorescence microscopy. Yeast nuclei

367

were stained with DAPI. RipI was observed to be exclusively localized in the nucleus

368

due to its co-staining with DAPI (Fig. 7). RipI

369

localization as the full-length protein , indicating that this fragment is not essential for

370

nuclear localization. On the other hand, the dispersion of the RipI

371

mutants in the whole cell indicated that the N-terminal 1-174aa was critical for

372

nuclear localization. Most importantly, this result also suggested that the predicted

373

integrase might be functional in yeast cells to instigate RipI-nucleus interaction.

374

Interestingly, the RipIR117G mutant is distinctly located in the nucleus, suggesting that

375

the role of R117 residue is rather relevant to the function of RipI than its localization.

mutant showed the same subcellular

RI PT

C

and RipI

ints

M AN U

SC

N

376

(The figure 7 file is larger than 10 MB and is uploaded separately)

378

Fig. 7. Subcellular co-localization of RipI and its mutated proteins in yeast cells.

379

Fresh cultures of wild type strain W303-1A transformed with pYX223-ripI,

380

pYX223-ripI

381

induced by galactose for 8 h followed by DAPI staining. RipI and its mutated proteins

382

were co-expressed with red fluorescence protein. DAPI-stained nuclei emit blue

383

fluorescence. Co-localization is seen in merged images (purple) which indicate

384

nuclear localization of the protein. Bar, 5 µm.

, pYX223-ripI

ints

, pYX223-ripIR117G and pYX223-ripI

C

, were

AC C

EP

N

TE D

377

385 386

Although RipI was found to be localized precisely in the nucleus, no typical

387

nuclear localization signal (NLS) (Dingwall and Laskey, 1986; Dingwall et al., 1988;

19

ACCEPTED MANUSCRIPT Kalderon et al., 1984; Shields and Yang, 1997) was identified in RipI sequence.

389

However, the fact that RipI without N-terminal segment lost the nuclear localization

390

implies that the N-terminal region of the protein probably contains a cryptic NLS (Gu

391

et al., 2003) or multiple NLS (Krauer et al., 2004; Theodore et al., 2008) necessitating

392

further studies.

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388

393

3.6 Genome-wide analysis of transcription of RipI-expressing yeast cells by

395

high-throughput RNA sequencing.

M AN U

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394

In eukaryotic cells including S. cerevisiae, apoptosis has always been closely

397

linked to DNA damage or other DNA inactivities (Burhans, 2003; Jazayeri et al.,

398

2004). The fact that RipI induces apoptosis and is located in the nucleus led us to

399

further characterize the downstream signaling pathway. Therefore, we performed

400

high-throughput RNA sequencing analysis of both W303-1A: pYES2-ripI and

401

W303-1A: pYES2/NTA strains. After

filtering

out

EP

402

TE D

396

the

low-quality

sequences

by

SeqPrep

(https://github.com/jstjohn/SeqPrep) and Sickle (https://github.com/najoshi/sickle), a

404

mean of around 41860912 clean reads, comprising 5795237469 nucleotides was

405

obtained in W303-1A: pYES2-ripI, while 50601395 clean reads, comprising

406

7063376511 nucleotides in W303-1A: pYES2/NTA. The Q20 and Q30 were

407

97.68±0.78% and 92.95±1.97% in W303-1A: pYES2-ripI, while 97.91±0.41% and

408

93.30±1.11% in W303-1A: pYES2/NTA, respectively. An overview of the

409

sequencing and assembly is shown in Table 1.

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403

20

ACCEPTED MANUSCRIPT 410

Table 1 Overview of Sequencing and Assembly among three samples of W303-1A:

RI PT

pYES2-ripI strain and three samples of W303-1A: pYES2/NTA strain. RipI-1

RipI-2

RipI-3

vector-1

vector-2

vector-3

Total reads Total nucleotides Error% Q20% Q30% Total mapped

55465430 7640645767 0.0125 97.82 93.23 46126213 (83.16% ) 2299626 ( 4.15% ) 43826587 (79.02% )

38453540 5221178803 0.0155 96.66 90.42 34377507 (89.40% ) 1099940 ( 2.86% ) 33277567 (86.54% )

31663768 4523887837 0.0106 98.56 95.21 27574999 (87.09% ) 1257988 ( 3.97% ) 26317011 (83.11% )

56891684 7776344819 0.0132 97.43 92.42 47629785 (83.72% ) 2254736 ( 3.96% ) 45375049 (79.76% )

52348364 7311011923 0.0127 97.87 92.62 42864358 (81.88% ) 2499242 ( 4.77% ) 40365116 (77.11% )

42564138 6102772790 0.0109 98.44 94.87 35693120 (83.86% ) 1867360 ( 4.39% ) 33825760 (79.47% )

Uniquely mapped

M AN U

Multiple mapped

SC

Samples

Q20, Q30%: Percentage of nucleotides with Phred score >20, 30;

411

TE D

Error%: Error percentage of nucleotides.

Subsequently, we used TopHat (http://tophat.cbcb.umd.edu/) (Trapnell et al.,

413

2009; Trapnell et al., 2012) to map the clean reads to the known genes in

414

Saccharomyces Genome Database (http://www.yeast genome. org). To guarantee the

415

highest possible data quality, the read pairs that aligned to more than one gene, or

416

contained two or more mismatches in their alignments were discarded. After this

417

filtering step, approximate 34473721 and 39855308 high-quality uniquely mapped

418

transcriptome reads of W303-1A: pYES2-ripI and W303-1A: pYES2/NTA were

419

retained. Mapping statistics are shown in line 8 to 10 of Table 1.

420

AC C

EP

412

Finally, 6304 unigenes were assembly yielded and the gene expression levels of

21

ACCEPTED MANUSCRIPT all the six samples were computed. Consecutively, the differentially expressed genes

422

in W303-1A: pYES2-ripI and W303-1A: pYES2/NTA were calculated by Cuffdiff

423

(http://cole-trapnell-lab.github.io/cufflinks/cuffdiff/index.html) (Trapnell et al., 2010).

424

Fig. 8A shows the distribution of logFC (RipI/vector) of the mean FPKM (fragments

425

per kilobase of exon per million fragments mapped) values for each yeast gene

426

(Reiner et al., 2003; Trapnell et al., 2013). Each spot represents a unigene. Left-side

427

spots represent the down-regulated unigenes and right-side spots represent the

428

up-regulated unigenes. Spots close to 0 represent the genes with low expression level.

M AN U

SC

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421

429

All the assembled unigenes were examined against the KEGG (Kyoto

430

Encyclopedia of Genes and Genomes) and GO (Gene Ontology) databases. In the

431

KEGG

432

(http://kobas.cbi.pku.edu.cn/home.do), 6304 unigenes were classified into 241 KEGG

433

pathways. However, only two relevant pathways were statistically significant with a

434

P-fdr (Bi and Liu, 2016) <0.05, i.e., ribosome and biosynthesis of amino acids (Fig.

435

8B). The highest number of differentially expressed genes was assigned to the

436

ribosome, wherein 101 unigenes exhibited lower expression level in W303-1A:

437

pYES2-ripI than W303-1A: pYES2/NTA, with a P-fdr <10-5.

analysis

(Xie

et

al.,

2011)

by

KOBAS

EP

TE D

enrichment

AC C

438

pathway

22

439

TE D

M AN U

SC

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ACCEPTED MANUSCRIPT

Fig. 8. High-throughput RNA sequencing analysis results. (A) Visualized

441

representation of differentially expressed genes between W303-1A: pYES2-ripI and

442

W303-1A: pYES2/NTA. Red and yellow spots represent the up-regulated genes with

443

P-fdr <0.05 and P-fdr <0.01. Blue and green spots represent the down-regulated genes

444

with P-fdr <0.05 and P-fdr <0.01. (B) Histogram presentation of KEGG. Vertical

445

coordinates represent the ratios of differentially expressed unigenes between

446

W303-1A: pYES2-ripI and W303-1A: pYES2/NTA in each pathway. *** represents

447

the P-fdr <0.001, * represents the P-fdr <0.05. (C) Histogram presentation of GO

AC C

EP

440

23

ACCEPTED MANUSCRIPT classification related to the nucleus. The results are summarized in three main

449

categories: biological process, cellular component and molecular function. Vertical

450

coordinates represent the ratios of differentially expressed unigenes between

451

W303-1A: pYES2-ripI and W303-1A: pYES2/NTA in each class. *** represents the

452

P-fdr <0.001, ** represents the P-fdr <0.01.

453

enrichment

analysis

(Bauer

et

al.,

2008)

SC

GO-term

454

RI PT

448

by

GOatools

(https://github.com/tanghaibao/GOatools) showed that, out of the 6304 unigenes,

456

1701 exhibited differential expression levels in W303-1A: pYES2-ripI as compared

457

with W303-1A: pYES2/NTA. Interestingly, 42 GO classes were found to be related to

458

the nucleus with a P-fdr <10-2 (Fig. 8C, Table S2), including retrotransposon

459

nucleocapsid,

460

ribonucleoprotein complex and DNA recombination. Five classes with remarkablely

461

high

462

RNA-mediated transposition (85.90%), RNA-directed DNA polymerase activity

463

(85.29%), DNA integration (79.25%), retrotransposon nucleocapsid (87.01%) and

464

RNA-DNA hybrid ribonuclease activity (78.72%), respectively.

nucleotide

TE D

binding,

differential

genes

were

binding,

particularly

RNA

interesting,

binding,

including

EP

of

acid

AC C

465

ratios

nucleic

M AN U

455

Taken together, these results indicated that localization of RipI in the nucleus

466

tremendously affects the expression of many genes, particularly those related to the

467

signaling pathway of the nucleus. Also, both large and small-subunit ribosome genes

468

were remarkably down-regulated, suggesting that the over-expression of RipI inhibits

469

protein synthesis. Finally, these results suggested that RipI triggers apoptotic cell

24

ACCEPTED MANUSCRIPT 470

death in yeast by the exertion of its integrase function, warranting further

471

investigation.

472

Acknowledgements

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473

We thank Prof. Hai-hong Wang at South China Agricultural University for

475

providing the GMI1000 strain. This work was partly supported by the National

476

Natural Science Foundation of China (J1310025).

M AN U

477

SC

474

478

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Hightlights  Phytopathogenic effector protein RipI is fatal toxic to Saccharomyces cerevisiae.  RipI induces apoptosis potentially dependent on its predicted integrase domain. 


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 RipI targets to yeast nucleus and reduces the expression levels of ribosomal genes.

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 RipI changes the expression levels of nucleus or recombination related genes.