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A Novel Pathogenesis-Related Class 10 Protein Gly m 4l, Increases Resistance upon Phytophthora sojae Infection in Soybean (Glycine max [L.] Merr.).

Fan S, Jiang L, Wu J, Dong L, Cheng Q, Xu P, Zhang S - PLoS ONE (2015)

Bottom Line: HQ913577.1).The recombinant Gly m 4l protein showed RNase activity and displayed directly antimicrobial activity that inhibited hyphal growth and reduced zoospore release in P. sojae.Further analyses showed that the RNase activity of the recombinant protein to degrading tRNA was significantly affected in the presence of zeatin.

View Article: PubMed Central - PubMed

Affiliation: Soybean Research Institute, Key Laboratory of Soybean Biology of Chinese Education Ministry, Northeast Agricultural University, Harbin, 150030, Heilongjiang, People's Republic of China.

ABSTRACT
Phytophthora root and stem rot of soybean, caused by Phytophthora sojae (P. sojae), is a destructive disease in many soybean planting regions worldwide. In a previous study, an expressed sequence tag (EST) homolog of the major allergen Pru ar 1 in apricot (Prunus armeniaca) was identified up-regulated in the highly resistant soybean 'Suinong 10' infected with P. sojae. Here, the full length of the EST was isolated using rapid amplification of cDNA ends (RACE). It showed the highest homology of 53.46% with Gly m 4 after comparison with the eight soybean allergen families reported and was named Gly m 4-like (Gly m 4l, GenBank accession no. HQ913577.1). The cDNA full length of Gly m 4l was 707 bp containing a 474 bp open reading frame encoding a polypeptide of 157 amino acids. Sequence analysis suggests that Gly m 4l contains a conserved 'P-loop' (phosphate-binding loop) motif at residues 47-55 aa and a Bet v 1 domain at residues 87-120 aa. The transcript abundance of Gly m 4l was significantly induced by P. sojae, salicylic acid (SA), NaCl, and also responded to methyl jasmonic acid (MeJA) and ethylene (ET). The recombinant Gly m 4l protein showed RNase activity and displayed directly antimicrobial activity that inhibited hyphal growth and reduced zoospore release in P. sojae. Further analyses showed that the RNase activity of the recombinant protein to degrading tRNA was significantly affected in the presence of zeatin. Over-expression of Gly m 4l in susceptible 'Dongnong 50' soybean showed enhanced resistance to P. sojae. These results indicated that Gly m 4l protein played an important role in the defense of soybean against P. sojae infection.

No MeSH data available.


Related in: MedlinePlus

RNase and DNase activities of the recombinant Gly m 4l protein.(A) RNase activity of Gly m 4l protein under different pH conditions. Samples containing 10 μg total soybean RNA were incubated with the presence of 10 μg Gly m 4l protein at 37°C for 0, 2 and 4 h at pH 3, 5, 7 and 9 from left to right. (B) RNase activity of the recombinant Gly m 4l protein analyzed in the presence of zeatin using total soybean RNA at pH 7. Samples containing 10 μg total soybean RNA were incubated with the presence of 10 μg Gly m 4l protein and 10 mM zeatin at 37°C for 30 min, 1, 2, 3 and 4 h. The samples incubated with the presence of the recombinant Gly m 4l protein or boiled Gly m 4l protein and zeatin were used as negative controls. (C) RNase activity of the recombinant Gly m 4l protein analyzed in the presence of zeatin using yeast tRNA. Samples containing 100 μg yeast tRNA were incubated with the presence of 10 μg recombinant Gly m 4l protein and 10 mM zeatin at 37°C for 30 min. The samples incubated with the presence of Gly m 4l protein alone or boiled Gly m 4l protein and zeatin were used as controls. The results were calculated by the 2−ΔΔCt method. (D) DNase activity of the recombinant Gly m 4l protein under different pH conditions. Samples containing 10 μg total soybean DNA and 10 μg Gly m 4l protein were incubated at 37°C for 0 and 4 h at pH 3, 5, 7 and 9 from left to right. The experiments were performed on three technical replicates and statistically analysed using Student’s t-test (*P<0.05, **P<0.01). Bars indicate standard error of the mean (SE).
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pone.0140364.g006: RNase and DNase activities of the recombinant Gly m 4l protein.(A) RNase activity of Gly m 4l protein under different pH conditions. Samples containing 10 μg total soybean RNA were incubated with the presence of 10 μg Gly m 4l protein at 37°C for 0, 2 and 4 h at pH 3, 5, 7 and 9 from left to right. (B) RNase activity of the recombinant Gly m 4l protein analyzed in the presence of zeatin using total soybean RNA at pH 7. Samples containing 10 μg total soybean RNA were incubated with the presence of 10 μg Gly m 4l protein and 10 mM zeatin at 37°C for 30 min, 1, 2, 3 and 4 h. The samples incubated with the presence of the recombinant Gly m 4l protein or boiled Gly m 4l protein and zeatin were used as negative controls. (C) RNase activity of the recombinant Gly m 4l protein analyzed in the presence of zeatin using yeast tRNA. Samples containing 100 μg yeast tRNA were incubated with the presence of 10 μg recombinant Gly m 4l protein and 10 mM zeatin at 37°C for 30 min. The samples incubated with the presence of Gly m 4l protein alone or boiled Gly m 4l protein and zeatin were used as controls. The results were calculated by the 2−ΔΔCt method. (D) DNase activity of the recombinant Gly m 4l protein under different pH conditions. Samples containing 10 μg total soybean DNA and 10 μg Gly m 4l protein were incubated at 37°C for 0 and 4 h at pH 3, 5, 7 and 9 from left to right. The experiments were performed on three technical replicates and statistically analysed using Student’s t-test (*P<0.05, **P<0.01). Bars indicate standard error of the mean (SE).

Mentions: To examine the RNase activity of the recombinant Gly m 4l protein, 10 μg total RNA from ‘Suinong 10’ soybean was incubated with 10 μg purified Gly m 4l protein at different pH conditions. As shown in Fig 6A, the RNase activities of Gly m 4l protein increased at 2 h and 4 h with the increase of pH from 3 to 7. At pH 9, almost no RNA digestion was detected at 2 h, while RNA was almost totally degraded at 4 h. To evaluate the effect of cytokinin on the RNase activity of the Gly m 4l protein, we examined the RNase activity in the presence of 10 mM zeatin at pH 7. The RNA digestion was delayed by 30 min in the presence of zeatin compared to that without zeatin (Fig 6B). Meanwhile, RNase activity of Gly m 4l was also checked using yeast tRNA, and the results showed that RNase activity of Gly m 4l towards yeast tRNA with the presence of zeatin was significantly lower than that without zeatin (Fig 6C).


A Novel Pathogenesis-Related Class 10 Protein Gly m 4l, Increases Resistance upon Phytophthora sojae Infection in Soybean (Glycine max [L.] Merr.).

Fan S, Jiang L, Wu J, Dong L, Cheng Q, Xu P, Zhang S - PLoS ONE (2015)

RNase and DNase activities of the recombinant Gly m 4l protein.(A) RNase activity of Gly m 4l protein under different pH conditions. Samples containing 10 μg total soybean RNA were incubated with the presence of 10 μg Gly m 4l protein at 37°C for 0, 2 and 4 h at pH 3, 5, 7 and 9 from left to right. (B) RNase activity of the recombinant Gly m 4l protein analyzed in the presence of zeatin using total soybean RNA at pH 7. Samples containing 10 μg total soybean RNA were incubated with the presence of 10 μg Gly m 4l protein and 10 mM zeatin at 37°C for 30 min, 1, 2, 3 and 4 h. The samples incubated with the presence of the recombinant Gly m 4l protein or boiled Gly m 4l protein and zeatin were used as negative controls. (C) RNase activity of the recombinant Gly m 4l protein analyzed in the presence of zeatin using yeast tRNA. Samples containing 100 μg yeast tRNA were incubated with the presence of 10 μg recombinant Gly m 4l protein and 10 mM zeatin at 37°C for 30 min. The samples incubated with the presence of Gly m 4l protein alone or boiled Gly m 4l protein and zeatin were used as controls. The results were calculated by the 2−ΔΔCt method. (D) DNase activity of the recombinant Gly m 4l protein under different pH conditions. Samples containing 10 μg total soybean DNA and 10 μg Gly m 4l protein were incubated at 37°C for 0 and 4 h at pH 3, 5, 7 and 9 from left to right. The experiments were performed on three technical replicates and statistically analysed using Student’s t-test (*P<0.05, **P<0.01). Bars indicate standard error of the mean (SE).
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Related In: Results  -  Collection

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pone.0140364.g006: RNase and DNase activities of the recombinant Gly m 4l protein.(A) RNase activity of Gly m 4l protein under different pH conditions. Samples containing 10 μg total soybean RNA were incubated with the presence of 10 μg Gly m 4l protein at 37°C for 0, 2 and 4 h at pH 3, 5, 7 and 9 from left to right. (B) RNase activity of the recombinant Gly m 4l protein analyzed in the presence of zeatin using total soybean RNA at pH 7. Samples containing 10 μg total soybean RNA were incubated with the presence of 10 μg Gly m 4l protein and 10 mM zeatin at 37°C for 30 min, 1, 2, 3 and 4 h. The samples incubated with the presence of the recombinant Gly m 4l protein or boiled Gly m 4l protein and zeatin were used as negative controls. (C) RNase activity of the recombinant Gly m 4l protein analyzed in the presence of zeatin using yeast tRNA. Samples containing 100 μg yeast tRNA were incubated with the presence of 10 μg recombinant Gly m 4l protein and 10 mM zeatin at 37°C for 30 min. The samples incubated with the presence of Gly m 4l protein alone or boiled Gly m 4l protein and zeatin were used as controls. The results were calculated by the 2−ΔΔCt method. (D) DNase activity of the recombinant Gly m 4l protein under different pH conditions. Samples containing 10 μg total soybean DNA and 10 μg Gly m 4l protein were incubated at 37°C for 0 and 4 h at pH 3, 5, 7 and 9 from left to right. The experiments were performed on three technical replicates and statistically analysed using Student’s t-test (*P<0.05, **P<0.01). Bars indicate standard error of the mean (SE).
Mentions: To examine the RNase activity of the recombinant Gly m 4l protein, 10 μg total RNA from ‘Suinong 10’ soybean was incubated with 10 μg purified Gly m 4l protein at different pH conditions. As shown in Fig 6A, the RNase activities of Gly m 4l protein increased at 2 h and 4 h with the increase of pH from 3 to 7. At pH 9, almost no RNA digestion was detected at 2 h, while RNA was almost totally degraded at 4 h. To evaluate the effect of cytokinin on the RNase activity of the Gly m 4l protein, we examined the RNase activity in the presence of 10 mM zeatin at pH 7. The RNA digestion was delayed by 30 min in the presence of zeatin compared to that without zeatin (Fig 6B). Meanwhile, RNase activity of Gly m 4l was also checked using yeast tRNA, and the results showed that RNase activity of Gly m 4l towards yeast tRNA with the presence of zeatin was significantly lower than that without zeatin (Fig 6C).

Bottom Line: HQ913577.1).The recombinant Gly m 4l protein showed RNase activity and displayed directly antimicrobial activity that inhibited hyphal growth and reduced zoospore release in P. sojae.Further analyses showed that the RNase activity of the recombinant protein to degrading tRNA was significantly affected in the presence of zeatin.

View Article: PubMed Central - PubMed

Affiliation: Soybean Research Institute, Key Laboratory of Soybean Biology of Chinese Education Ministry, Northeast Agricultural University, Harbin, 150030, Heilongjiang, People's Republic of China.

ABSTRACT
Phytophthora root and stem rot of soybean, caused by Phytophthora sojae (P. sojae), is a destructive disease in many soybean planting regions worldwide. In a previous study, an expressed sequence tag (EST) homolog of the major allergen Pru ar 1 in apricot (Prunus armeniaca) was identified up-regulated in the highly resistant soybean 'Suinong 10' infected with P. sojae. Here, the full length of the EST was isolated using rapid amplification of cDNA ends (RACE). It showed the highest homology of 53.46% with Gly m 4 after comparison with the eight soybean allergen families reported and was named Gly m 4-like (Gly m 4l, GenBank accession no. HQ913577.1). The cDNA full length of Gly m 4l was 707 bp containing a 474 bp open reading frame encoding a polypeptide of 157 amino acids. Sequence analysis suggests that Gly m 4l contains a conserved 'P-loop' (phosphate-binding loop) motif at residues 47-55 aa and a Bet v 1 domain at residues 87-120 aa. The transcript abundance of Gly m 4l was significantly induced by P. sojae, salicylic acid (SA), NaCl, and also responded to methyl jasmonic acid (MeJA) and ethylene (ET). The recombinant Gly m 4l protein showed RNase activity and displayed directly antimicrobial activity that inhibited hyphal growth and reduced zoospore release in P. sojae. Further analyses showed that the RNase activity of the recombinant protein to degrading tRNA was significantly affected in the presence of zeatin. Over-expression of Gly m 4l in susceptible 'Dongnong 50' soybean showed enhanced resistance to P. sojae. These results indicated that Gly m 4l protein played an important role in the defense of soybean against P. sojae infection.

No MeSH data available.


Related in: MedlinePlus