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Cysteine protease enhances plant-mediated bollworm RNA interference.

Mao YB, Xue XY, Tao XY, Yang CQ, Wang LJ, Chen XY - Plant Mol. Biol. (2013)

Bottom Line: Furthermore, the pre-fed larvae exhibited enhanced RNAi effects after ingestion of the dsRNA-expressing plant.We found that cotton plants harboring both 35S:dsCYP6AE14 and 35S:GhCP1 were better protected from bollworm than either of the single-transgene lines.Our results demonstrate that plant cysteine proteases, which have the activity of increasing PM permeability, can be used to improve the plant-mediated RNAi against herbivorous insects.

View Article: PubMed Central - PubMed

Affiliation: National Key Laboratory of Plant Molecular Genetics, National Plant Gene Research Center, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 300 Fenglin Road, Shanghai 200032, People's Republic of China.

ABSTRACT
Oral ingestion of plant-expressed double stranded RNA (dsRNA) triggers target gene suppression in insect. An important step of this process is the transmission of dsRNA from plant to midgut cells. Insect peritrophic matrix (PM) presents a barrier that prevents large molecules from entering midgut cells. Here, we show that uptake of plant cysteine proteases, such as GhCP1 from cotton (Gossypium hirsutum) and AtCP2 from Arabidopsis, by cotton bollworm (Helicoverpa armigera) larvae resulted in attenuating the PM. When GhCP1 or AtCP2 pre-fed larvae were transferred to gossypol-containing diet, the bollworm accumulated higher content of gossypol in midgut. Larvae previously ingested GhCP1 or AtCP2 were more susceptible to infection by Dendrolimus punctatus cytoplasmic polyhedrosis virus (DpCPV), a dsRNA virus. Furthermore, the pre-fed larvae exhibited enhanced RNAi effects after ingestion of the dsRNA-expressing plant. The bollworm P450 gene CYP6AE14 is involved in the larval tolerance to gossypol; cotton plants producing dsRNA of CYP6AE14 (dsCYP6AE14) were more resistant to bollworm feeding (Mao et al. in Transgenic Res 20:665-673, 2011). We found that cotton plants harboring both 35S:dsCYP6AE14 and 35S:GhCP1 were better protected from bollworm than either of the single-transgene lines. Our results demonstrate that plant cysteine proteases, which have the activity of increasing PM permeability, can be used to improve the plant-mediated RNAi against herbivorous insects.

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Expression patterns of GhCP1 and AtCP2. aGhCP1 expression in cotton (G. hirsutum). Cotyledon of 1-week-old seedlings, and leaf (the second leaf from top), petal and ovule (day 3 post-anthesis) of adult plant (12 weeks old) were analyzed; bAtCP2 expression in leaf, stem, inflorescence and silique of 4-week-old plants of A. thaliana; Expression of GhCP1 (c) and AtCP2 (d) after wounding. The 4–week-old cotton plant and the Arabidopsis leaves were treated by wounding for indicated times. The expressions of GhCP1 in cotyledon and AtCP2 in leaf were set to 1. In wounding treatment, the expression level just before treatments was set to 1. Each investigation had at least three biological repeats, error bars represent standard deviation (SD)
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Fig1: Expression patterns of GhCP1 and AtCP2. aGhCP1 expression in cotton (G. hirsutum). Cotyledon of 1-week-old seedlings, and leaf (the second leaf from top), petal and ovule (day 3 post-anthesis) of adult plant (12 weeks old) were analyzed; bAtCP2 expression in leaf, stem, inflorescence and silique of 4-week-old plants of A. thaliana; Expression of GhCP1 (c) and AtCP2 (d) after wounding. The 4–week-old cotton plant and the Arabidopsis leaves were treated by wounding for indicated times. The expressions of GhCP1 in cotyledon and AtCP2 in leaf were set to 1. In wounding treatment, the expression level just before treatments was set to 1. Each investigation had at least three biological repeats, error bars represent standard deviation (SD)

Mentions: To isolate cysteine proteases from cotton (G. hirsutum), we searched database for genes encoding proteins with sequence similarities to MirCP1 (AF019145), a cysteine protease from maize (Pechan et al. 2002). Three of them, GhCP1 (CAE54307), GhCP2 (AY171099) and GhCP3 (CAE54306) were identified, and they show 48, 37 and 28 % amino acid sequence identities, respectively, to MirCP1. Analysis with RT-PCR and quantitative RT-PCR (qRT-PCR) showed that GhCP1 had a high level of expression in petal only, and GhCP3 was also highly expressed in petal, with a low level of transcripts present in leaf, cotyledon and ovule, whereas GhCP2 was expressed widely in cotton plants (Fig. 1a; Fig. S1). In A. thaliana genome, AtCP1 (At4g11310) and AtCP2 (At4g11320) encode putative cysteine proteases and share high sequence amino acid sequence identities (45 and 44 %, respectively) to MirCP1. Although located side by side in genomic locus, AtCP1 and AtCP2 exhibited different expression patterns. AtCP1 was expressed exclusively in inflorescence (Fig. S2), whereas AtCP2 was highly expressed in seedling, root and inflorescence, with a low level of expression also detected in stem (Fig. 1b; Fig. S2).Fig. 1


Cysteine protease enhances plant-mediated bollworm RNA interference.

Mao YB, Xue XY, Tao XY, Yang CQ, Wang LJ, Chen XY - Plant Mol. Biol. (2013)

Expression patterns of GhCP1 and AtCP2. aGhCP1 expression in cotton (G. hirsutum). Cotyledon of 1-week-old seedlings, and leaf (the second leaf from top), petal and ovule (day 3 post-anthesis) of adult plant (12 weeks old) were analyzed; bAtCP2 expression in leaf, stem, inflorescence and silique of 4-week-old plants of A. thaliana; Expression of GhCP1 (c) and AtCP2 (d) after wounding. The 4–week-old cotton plant and the Arabidopsis leaves were treated by wounding for indicated times. The expressions of GhCP1 in cotyledon and AtCP2 in leaf were set to 1. In wounding treatment, the expression level just before treatments was set to 1. Each investigation had at least three biological repeats, error bars represent standard deviation (SD)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3755213&req=5

Fig1: Expression patterns of GhCP1 and AtCP2. aGhCP1 expression in cotton (G. hirsutum). Cotyledon of 1-week-old seedlings, and leaf (the second leaf from top), petal and ovule (day 3 post-anthesis) of adult plant (12 weeks old) were analyzed; bAtCP2 expression in leaf, stem, inflorescence and silique of 4-week-old plants of A. thaliana; Expression of GhCP1 (c) and AtCP2 (d) after wounding. The 4–week-old cotton plant and the Arabidopsis leaves were treated by wounding for indicated times. The expressions of GhCP1 in cotyledon and AtCP2 in leaf were set to 1. In wounding treatment, the expression level just before treatments was set to 1. Each investigation had at least three biological repeats, error bars represent standard deviation (SD)
Mentions: To isolate cysteine proteases from cotton (G. hirsutum), we searched database for genes encoding proteins with sequence similarities to MirCP1 (AF019145), a cysteine protease from maize (Pechan et al. 2002). Three of them, GhCP1 (CAE54307), GhCP2 (AY171099) and GhCP3 (CAE54306) were identified, and they show 48, 37 and 28 % amino acid sequence identities, respectively, to MirCP1. Analysis with RT-PCR and quantitative RT-PCR (qRT-PCR) showed that GhCP1 had a high level of expression in petal only, and GhCP3 was also highly expressed in petal, with a low level of transcripts present in leaf, cotyledon and ovule, whereas GhCP2 was expressed widely in cotton plants (Fig. 1a; Fig. S1). In A. thaliana genome, AtCP1 (At4g11310) and AtCP2 (At4g11320) encode putative cysteine proteases and share high sequence amino acid sequence identities (45 and 44 %, respectively) to MirCP1. Although located side by side in genomic locus, AtCP1 and AtCP2 exhibited different expression patterns. AtCP1 was expressed exclusively in inflorescence (Fig. S2), whereas AtCP2 was highly expressed in seedling, root and inflorescence, with a low level of expression also detected in stem (Fig. 1b; Fig. S2).Fig. 1

Bottom Line: Furthermore, the pre-fed larvae exhibited enhanced RNAi effects after ingestion of the dsRNA-expressing plant.We found that cotton plants harboring both 35S:dsCYP6AE14 and 35S:GhCP1 were better protected from bollworm than either of the single-transgene lines.Our results demonstrate that plant cysteine proteases, which have the activity of increasing PM permeability, can be used to improve the plant-mediated RNAi against herbivorous insects.

View Article: PubMed Central - PubMed

Affiliation: National Key Laboratory of Plant Molecular Genetics, National Plant Gene Research Center, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 300 Fenglin Road, Shanghai 200032, People's Republic of China.

ABSTRACT
Oral ingestion of plant-expressed double stranded RNA (dsRNA) triggers target gene suppression in insect. An important step of this process is the transmission of dsRNA from plant to midgut cells. Insect peritrophic matrix (PM) presents a barrier that prevents large molecules from entering midgut cells. Here, we show that uptake of plant cysteine proteases, such as GhCP1 from cotton (Gossypium hirsutum) and AtCP2 from Arabidopsis, by cotton bollworm (Helicoverpa armigera) larvae resulted in attenuating the PM. When GhCP1 or AtCP2 pre-fed larvae were transferred to gossypol-containing diet, the bollworm accumulated higher content of gossypol in midgut. Larvae previously ingested GhCP1 or AtCP2 were more susceptible to infection by Dendrolimus punctatus cytoplasmic polyhedrosis virus (DpCPV), a dsRNA virus. Furthermore, the pre-fed larvae exhibited enhanced RNAi effects after ingestion of the dsRNA-expressing plant. The bollworm P450 gene CYP6AE14 is involved in the larval tolerance to gossypol; cotton plants producing dsRNA of CYP6AE14 (dsCYP6AE14) were more resistant to bollworm feeding (Mao et al. in Transgenic Res 20:665-673, 2011). We found that cotton plants harboring both 35S:dsCYP6AE14 and 35S:GhCP1 were better protected from bollworm than either of the single-transgene lines. Our results demonstrate that plant cysteine proteases, which have the activity of increasing PM permeability, can be used to improve the plant-mediated RNAi against herbivorous insects.

Show MeSH
Related in: MedlinePlus