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Co-silencing of tomato S-adenosylhomocysteine hydrolase genes confers increased immunity against Pseudomonas syringae pv. tomato DC3000 and enhanced tolerance to drought stress.

Li X, Huang L, Hong Y, Zhang Y, Liu S, Li D, Zhang H, Song F - Front Plant Sci (2015)

Bottom Line: Virus-induced gene silencing-based knockdown of individual SlSAHH gene did not affect the growth performance and the response to Pst DC3000.The SlSAHH-co-silenced plants displayed increased resistance to Pst DC3000 but did not alter the resistance to B. cinerea.Co-silencing of SlSAHHs resulted in constitutively activated defense responses including elevated SA level, upregulated expression of defense-related and PAMP-triggered immunity marker genes and increased callose deposition and H2O2 accumulation.

View Article: PubMed Central - PubMed

Affiliation: National Key Laboratory for Rice Biology, Institute of Biotechnology, Zhejiang University Hangzhou, China.

ABSTRACT
S-adenosylhomocysteine hydrolase (SAHH), catalyzing the reversible hydrolysis of S-adenosylhomocysteine (SAH) to adenosine and homocysteine, is a key enzyme that maintain the cellular methylation potential in all organisms. We report here the biological functions of tomato SlSAHHs in stress response. The tomato genome contains three SlSAHH genes that encode SlSAHH proteins with high level of sequence identity. qRT-PCR analysis revealed that SlSAHHs responded with distinct expression induction patterns to Pseudomonas syringae pv. tomato (Pst) DC3000 and Botrytis cinerea as well as to defense signaling hormones such as salicylic acid, jasmonic acid and a precursor of ethylene. Virus-induced gene silencing-based knockdown of individual SlSAHH gene did not affect the growth performance and the response to Pst DC3000. However, co-silencing of three SlSAHH genes using a conserved sequence led to significant inhibition of vegetable growth. The SlSAHH-co-silenced plants displayed increased resistance to Pst DC3000 but did not alter the resistance to B. cinerea. Co-silencing of SlSAHHs resulted in constitutively activated defense responses including elevated SA level, upregulated expression of defense-related and PAMP-triggered immunity marker genes and increased callose deposition and H2O2 accumulation. Furthermore, the SlSAHH-co-silenced plants also exhibited enhanced drought stress tolerance although they had relatively small roots. These data demonstrate that, in addition to the functions in growth and development, SAHHs also play important roles in regulating biotic and abiotic stress responses in plants.

No MeSH data available.


Related in: MedlinePlus

Virus-induced gene silencing-based silencing efficiency and specificity for SlSAHHs and co-silencing of SlSAHHs resulted in abnormal growth phenotypes. Alignment (A) and the identity and divergence (B) of the VIGS sequences from SlSAHHs for co-silencing. The VIGS sequences were aligned by ClustalW program in DNAStar software and the identity and divergence percentages were obtained from the alignment. (C) Silencing efficiency and specificity. Ten-day-old seedlings were infiltrated with agrobacteria carrying TRV-SlSAHH1, TRV-SlSAHH2, TRV-SlSAHH3, TRV-SlSAHHa or TRV-GUS constructs and leaf samples were collected 3 weeks after agroinfiltration. Transcript levels for SlSAHH genes were analyzed by qRT-PCR using a tomato SlActin gene as an internal control. (D) Co-silencing of SlSAHHs inhibited vegetable growth in TRV-SlSAHHa-infiltrated plants. Upper row, seedlings at the time of agroinfiltration; Lower row, growth performance of the agroinfiltrated plants at 5 weeks after agroinfiltration. The experiments were repeated twice with similar results. (E) and (F) Plant heights and whole plant weights of the TRV-SlSAHH1-, TRV-SlSAHH2-, TRV-SlSAHH3-, TRV-SlSAHHa-, or TRV-GUS-infiltrated plants. Six-week-old plants were collected to measure the heights and weights at weeks after agroinfiltration. Data presented are the means ± SD from three independent experiments and ∗ above the columns indicate significant differences at p < 0.05 level between the expression levels of SlSAHH genes in SlSAHH-silenced and TRV-GUS-infiltrated non-silenced plants (C) or between the plant heights and weights in TRV-GUS- and TRV-SlSAHHa-infiltrated plants (E) and (F).
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Figure 2: Virus-induced gene silencing-based silencing efficiency and specificity for SlSAHHs and co-silencing of SlSAHHs resulted in abnormal growth phenotypes. Alignment (A) and the identity and divergence (B) of the VIGS sequences from SlSAHHs for co-silencing. The VIGS sequences were aligned by ClustalW program in DNAStar software and the identity and divergence percentages were obtained from the alignment. (C) Silencing efficiency and specificity. Ten-day-old seedlings were infiltrated with agrobacteria carrying TRV-SlSAHH1, TRV-SlSAHH2, TRV-SlSAHH3, TRV-SlSAHHa or TRV-GUS constructs and leaf samples were collected 3 weeks after agroinfiltration. Transcript levels for SlSAHH genes were analyzed by qRT-PCR using a tomato SlActin gene as an internal control. (D) Co-silencing of SlSAHHs inhibited vegetable growth in TRV-SlSAHHa-infiltrated plants. Upper row, seedlings at the time of agroinfiltration; Lower row, growth performance of the agroinfiltrated plants at 5 weeks after agroinfiltration. The experiments were repeated twice with similar results. (E) and (F) Plant heights and whole plant weights of the TRV-SlSAHH1-, TRV-SlSAHH2-, TRV-SlSAHH3-, TRV-SlSAHHa-, or TRV-GUS-infiltrated plants. Six-week-old plants were collected to measure the heights and weights at weeks after agroinfiltration. Data presented are the means ± SD from three independent experiments and ∗ above the columns indicate significant differences at p < 0.05 level between the expression levels of SlSAHH genes in SlSAHH-silenced and TRV-GUS-infiltrated non-silenced plants (C) or between the plant heights and weights in TRV-GUS- and TRV-SlSAHHa-infiltrated plants (E) and (F).

Mentions: To understand the biological functions of SlSAHHs, a series of VIGS-based functional analyses was carried out. For this purpose, specific fragments for SlSAHH1, SlSAHH2, and SlSAHH3 were used to silence each of the individual SlSAHH genes. Considering that the SlSAHHs are highly conserved in amino acid sequences, a conserved fragment with high level of sequence identity among SlSAHHs (Figure 2A), designated as SlSAHHa, was also used to co-silence all SlSAHH genes. This SlSAHHa fragment was amplified from SlSAHH1 and showed >82% of sequence identity to the corresponding regions of SlSAHH2 and SlSAHH3 (Figure 2B). The silencing efficiency and specificity were estimated by qRT-PCR analysis of the transcript levels of each SlSAHH gene at 3 weeks after VIGS infiltration when >90% of the PDS-silenced plants displayed bleaching symptom. In SlSAHH1-, SlSAHH2-, and SlSAHH3-silenced plants, the transcript levels of SlSAHH1, SlSAHH2, and SlSAHH3 were decreased by 63, 65, and 65%, respectively, as compared with those in TRV-GUS-infiltrated non-silenced plants (Figure 2C). The expression levels of SlSAHH2 and SlSAHH3 in SlSAHH2-silenced plants were slightly increased (Figure 2C). However, the expression levels of SlSAHH3 in SlSAHH2-silenced plants and SlSAHH2 in SlSAHH3-silenced plants were upregulated by 2.6 and 2.3 folds, respectively, as compared with those in TRV-GUS-infiltrated non-silenced plants; while the expression of SlSAHH1 in SlSAHH2- and SlSAHH3-silenced plants was not significantly affected (Figure 2C). These data imply that SlSAHH2 and SlSAHH3 may have functional redundancy. In the TRV-SlSAHHa-infiltrated plants, the expression levels of SlSAHH1, SlSAHH2, and SlSAHH3 were simultaneously and significantly decreased by 68, 63, and 64%, respectively, as compared with those in TRV-GUS-infiltrated non-silenced plants (Figure 2C), indicating that SlSAHHa could co-suppress the expression of the SlSAHH genes in tomato.


Co-silencing of tomato S-adenosylhomocysteine hydrolase genes confers increased immunity against Pseudomonas syringae pv. tomato DC3000 and enhanced tolerance to drought stress.

Li X, Huang L, Hong Y, Zhang Y, Liu S, Li D, Zhang H, Song F - Front Plant Sci (2015)

Virus-induced gene silencing-based silencing efficiency and specificity for SlSAHHs and co-silencing of SlSAHHs resulted in abnormal growth phenotypes. Alignment (A) and the identity and divergence (B) of the VIGS sequences from SlSAHHs for co-silencing. The VIGS sequences were aligned by ClustalW program in DNAStar software and the identity and divergence percentages were obtained from the alignment. (C) Silencing efficiency and specificity. Ten-day-old seedlings were infiltrated with agrobacteria carrying TRV-SlSAHH1, TRV-SlSAHH2, TRV-SlSAHH3, TRV-SlSAHHa or TRV-GUS constructs and leaf samples were collected 3 weeks after agroinfiltration. Transcript levels for SlSAHH genes were analyzed by qRT-PCR using a tomato SlActin gene as an internal control. (D) Co-silencing of SlSAHHs inhibited vegetable growth in TRV-SlSAHHa-infiltrated plants. Upper row, seedlings at the time of agroinfiltration; Lower row, growth performance of the agroinfiltrated plants at 5 weeks after agroinfiltration. The experiments were repeated twice with similar results. (E) and (F) Plant heights and whole plant weights of the TRV-SlSAHH1-, TRV-SlSAHH2-, TRV-SlSAHH3-, TRV-SlSAHHa-, or TRV-GUS-infiltrated plants. Six-week-old plants were collected to measure the heights and weights at weeks after agroinfiltration. Data presented are the means ± SD from three independent experiments and ∗ above the columns indicate significant differences at p < 0.05 level between the expression levels of SlSAHH genes in SlSAHH-silenced and TRV-GUS-infiltrated non-silenced plants (C) or between the plant heights and weights in TRV-GUS- and TRV-SlSAHHa-infiltrated plants (E) and (F).
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Figure 2: Virus-induced gene silencing-based silencing efficiency and specificity for SlSAHHs and co-silencing of SlSAHHs resulted in abnormal growth phenotypes. Alignment (A) and the identity and divergence (B) of the VIGS sequences from SlSAHHs for co-silencing. The VIGS sequences were aligned by ClustalW program in DNAStar software and the identity and divergence percentages were obtained from the alignment. (C) Silencing efficiency and specificity. Ten-day-old seedlings were infiltrated with agrobacteria carrying TRV-SlSAHH1, TRV-SlSAHH2, TRV-SlSAHH3, TRV-SlSAHHa or TRV-GUS constructs and leaf samples were collected 3 weeks after agroinfiltration. Transcript levels for SlSAHH genes were analyzed by qRT-PCR using a tomato SlActin gene as an internal control. (D) Co-silencing of SlSAHHs inhibited vegetable growth in TRV-SlSAHHa-infiltrated plants. Upper row, seedlings at the time of agroinfiltration; Lower row, growth performance of the agroinfiltrated plants at 5 weeks after agroinfiltration. The experiments were repeated twice with similar results. (E) and (F) Plant heights and whole plant weights of the TRV-SlSAHH1-, TRV-SlSAHH2-, TRV-SlSAHH3-, TRV-SlSAHHa-, or TRV-GUS-infiltrated plants. Six-week-old plants were collected to measure the heights and weights at weeks after agroinfiltration. Data presented are the means ± SD from three independent experiments and ∗ above the columns indicate significant differences at p < 0.05 level between the expression levels of SlSAHH genes in SlSAHH-silenced and TRV-GUS-infiltrated non-silenced plants (C) or between the plant heights and weights in TRV-GUS- and TRV-SlSAHHa-infiltrated plants (E) and (F).
Mentions: To understand the biological functions of SlSAHHs, a series of VIGS-based functional analyses was carried out. For this purpose, specific fragments for SlSAHH1, SlSAHH2, and SlSAHH3 were used to silence each of the individual SlSAHH genes. Considering that the SlSAHHs are highly conserved in amino acid sequences, a conserved fragment with high level of sequence identity among SlSAHHs (Figure 2A), designated as SlSAHHa, was also used to co-silence all SlSAHH genes. This SlSAHHa fragment was amplified from SlSAHH1 and showed >82% of sequence identity to the corresponding regions of SlSAHH2 and SlSAHH3 (Figure 2B). The silencing efficiency and specificity were estimated by qRT-PCR analysis of the transcript levels of each SlSAHH gene at 3 weeks after VIGS infiltration when >90% of the PDS-silenced plants displayed bleaching symptom. In SlSAHH1-, SlSAHH2-, and SlSAHH3-silenced plants, the transcript levels of SlSAHH1, SlSAHH2, and SlSAHH3 were decreased by 63, 65, and 65%, respectively, as compared with those in TRV-GUS-infiltrated non-silenced plants (Figure 2C). The expression levels of SlSAHH2 and SlSAHH3 in SlSAHH2-silenced plants were slightly increased (Figure 2C). However, the expression levels of SlSAHH3 in SlSAHH2-silenced plants and SlSAHH2 in SlSAHH3-silenced plants were upregulated by 2.6 and 2.3 folds, respectively, as compared with those in TRV-GUS-infiltrated non-silenced plants; while the expression of SlSAHH1 in SlSAHH2- and SlSAHH3-silenced plants was not significantly affected (Figure 2C). These data imply that SlSAHH2 and SlSAHH3 may have functional redundancy. In the TRV-SlSAHHa-infiltrated plants, the expression levels of SlSAHH1, SlSAHH2, and SlSAHH3 were simultaneously and significantly decreased by 68, 63, and 64%, respectively, as compared with those in TRV-GUS-infiltrated non-silenced plants (Figure 2C), indicating that SlSAHHa could co-suppress the expression of the SlSAHH genes in tomato.

Bottom Line: Virus-induced gene silencing-based knockdown of individual SlSAHH gene did not affect the growth performance and the response to Pst DC3000.The SlSAHH-co-silenced plants displayed increased resistance to Pst DC3000 but did not alter the resistance to B. cinerea.Co-silencing of SlSAHHs resulted in constitutively activated defense responses including elevated SA level, upregulated expression of defense-related and PAMP-triggered immunity marker genes and increased callose deposition and H2O2 accumulation.

View Article: PubMed Central - PubMed

Affiliation: National Key Laboratory for Rice Biology, Institute of Biotechnology, Zhejiang University Hangzhou, China.

ABSTRACT
S-adenosylhomocysteine hydrolase (SAHH), catalyzing the reversible hydrolysis of S-adenosylhomocysteine (SAH) to adenosine and homocysteine, is a key enzyme that maintain the cellular methylation potential in all organisms. We report here the biological functions of tomato SlSAHHs in stress response. The tomato genome contains three SlSAHH genes that encode SlSAHH proteins with high level of sequence identity. qRT-PCR analysis revealed that SlSAHHs responded with distinct expression induction patterns to Pseudomonas syringae pv. tomato (Pst) DC3000 and Botrytis cinerea as well as to defense signaling hormones such as salicylic acid, jasmonic acid and a precursor of ethylene. Virus-induced gene silencing-based knockdown of individual SlSAHH gene did not affect the growth performance and the response to Pst DC3000. However, co-silencing of three SlSAHH genes using a conserved sequence led to significant inhibition of vegetable growth. The SlSAHH-co-silenced plants displayed increased resistance to Pst DC3000 but did not alter the resistance to B. cinerea. Co-silencing of SlSAHHs resulted in constitutively activated defense responses including elevated SA level, upregulated expression of defense-related and PAMP-triggered immunity marker genes and increased callose deposition and H2O2 accumulation. Furthermore, the SlSAHH-co-silenced plants also exhibited enhanced drought stress tolerance although they had relatively small roots. These data demonstrate that, in addition to the functions in growth and development, SAHHs also play important roles in regulating biotic and abiotic stress responses in plants.

No MeSH data available.


Related in: MedlinePlus