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Arabidopsis ALA1 and ALA2 Mediate RNAi-Based Antiviral Immunity

View Article: PubMed Central - PubMed

ABSTRACT

RNA intereferencing (RNAi) pathway regulates antiviral immunity and mediates plant growth and development. Despite considerable research efforts, a few components in RNAi pathway have been revealed, including ARGONAUTEs (AGOs), DICER-LIKEs (DCLs), RNA-dependent RNA polymerase 1 and 6 (RDR1/6), and ALTERED MERISTEM PROGRAM 1 (AMP1). In this study, we performed a forward genetic screening for enhancers of rdr6 via inoculation of CMV2aTΔ2b, a 2b-deficient Cucumber Mosaic Virus that is unable to suppress RNAi-mediated antiviral immunity. We uncover that the membrane-localized flippase Aminophospholipid ATPase 1 (ALA1) cooperates with RDR6 and RDR1 to promote antiviral immunity and regulate fertility in Arabidopsis. Moreover, we find that ALA2, a homolog of ALA1, also participates in antiviral immunity. Our findings suggest that ALA1 and ALA2 act as novel components in the RNAi pathway and function additively with RDR1 and RDR6 to mediate RNAi-based antiviral immunity and plant development.

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Analysis of ALA members in antiviral silencing. (A) Quantitative real-time PCR showed the relative expression levels of ALA family members in Col-0 and ala1-2 after inoculation with mock or CMV2aTΔ2b. The data are means (±SE) from three biological repeats. (B) Phenotypes of the ala3, ala7 and ala10 mutants at 21 days after infection with mock or CMV2aTΔ2b.
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Figure 6: Analysis of ALA members in antiviral silencing. (A) Quantitative real-time PCR showed the relative expression levels of ALA family members in Col-0 and ala1-2 after inoculation with mock or CMV2aTΔ2b. The data are means (±SE) from three biological repeats. (B) Phenotypes of the ala3, ala7 and ala10 mutants at 21 days after infection with mock or CMV2aTΔ2b.

Mentions: For Figure 6A, the expression of ALA family members was analyzed in Col-0 and ala1-2 at 21 days after inoculation with mock or CMV2aTΔ2b. For Supplementary Figure 4, the accumulation of genomic RNA of CMV2aTΔ2b was analyzed in Col-0 and ala1-2 at 21 days after CMV2aTΔ2b inoculation. The primers used for RNA detection of CMV2aTΔ2b were designed based on the conserved sequences from genomic RNA1 to RNA3 in the 3 prime end. The materials were harvested for RNA extraction using trizol (TRANSGENE, Cat.ET101-01), and reverse transcription was performed according to the kit (TRANSGENE, Cat. AT311-03). Quantitative real-time PCR was performed with EvaGreen 2∗qPCR MasterMix-Low ROX reagents (ABM, Cat. Mastermix-LR) using the ABI7500 real-time PCR system. ACTIN8 was used as the internal control. All of the experiments were repeated at least three biological times. Primers used for quantitative real-time PCR analysis are listed in Supplementary Table 1.


Arabidopsis ALA1 and ALA2 Mediate RNAi-Based Antiviral Immunity
Analysis of ALA members in antiviral silencing. (A) Quantitative real-time PCR showed the relative expression levels of ALA family members in Col-0 and ala1-2 after inoculation with mock or CMV2aTΔ2b. The data are means (±SE) from three biological repeats. (B) Phenotypes of the ala3, ala7 and ala10 mutants at 21 days after infection with mock or CMV2aTΔ2b.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5383662&req=5

Figure 6: Analysis of ALA members in antiviral silencing. (A) Quantitative real-time PCR showed the relative expression levels of ALA family members in Col-0 and ala1-2 after inoculation with mock or CMV2aTΔ2b. The data are means (±SE) from three biological repeats. (B) Phenotypes of the ala3, ala7 and ala10 mutants at 21 days after infection with mock or CMV2aTΔ2b.
Mentions: For Figure 6A, the expression of ALA family members was analyzed in Col-0 and ala1-2 at 21 days after inoculation with mock or CMV2aTΔ2b. For Supplementary Figure 4, the accumulation of genomic RNA of CMV2aTΔ2b was analyzed in Col-0 and ala1-2 at 21 days after CMV2aTΔ2b inoculation. The primers used for RNA detection of CMV2aTΔ2b were designed based on the conserved sequences from genomic RNA1 to RNA3 in the 3 prime end. The materials were harvested for RNA extraction using trizol (TRANSGENE, Cat.ET101-01), and reverse transcription was performed according to the kit (TRANSGENE, Cat. AT311-03). Quantitative real-time PCR was performed with EvaGreen 2∗qPCR MasterMix-Low ROX reagents (ABM, Cat. Mastermix-LR) using the ABI7500 real-time PCR system. ACTIN8 was used as the internal control. All of the experiments were repeated at least three biological times. Primers used for quantitative real-time PCR analysis are listed in Supplementary Table 1.

View Article: PubMed Central - PubMed

ABSTRACT

RNA intereferencing (RNAi) pathway regulates antiviral immunity and mediates plant growth and development. Despite considerable research efforts, a few components in RNAi pathway have been revealed, including ARGONAUTEs (AGOs), DICER-LIKEs (DCLs), RNA-dependent RNA polymerase 1 and 6 (RDR1/6), and ALTERED MERISTEM PROGRAM 1 (AMP1). In this study, we performed a forward genetic screening for enhancers of rdr6 via inoculation of CMV2aTΔ2b, a 2b-deficient Cucumber Mosaic Virus that is unable to suppress RNAi-mediated antiviral immunity. We uncover that the membrane-localized flippase Aminophospholipid ATPase 1 (ALA1) cooperates with RDR6 and RDR1 to promote antiviral immunity and regulate fertility in Arabidopsis. Moreover, we find that ALA2, a homolog of ALA1, also participates in antiviral immunity. Our findings suggest that ALA1 and ALA2 act as novel components in the RNAi pathway and function additively with RDR1 and RDR6 to mediate RNAi-based antiviral immunity and plant development.

No MeSH data available.


Related in: MedlinePlus