Santana L S, Costa M G C, Pirovani N M, Almeida A F, Alvim F C, Pirovani C P
Centro de Biotecnologia e Genética, Universidade Estadual de Santa Cruz, Inhéus, BA, Brasil.
Centro de Biotecnologia e Genética, Universidade Estadual de Santa Cruz, Inhéus, BA, Brasil
Genet Mol Res. 2014 Sep 26;13(3):7636-48. doi: 10.4238/2014.September.26.1.
In Brazil, most cocoa bean production occurs in Southern Bahia. Witches' broom disease arrived in this area in 1989 and has since caused heavy losses in production. The disease is caused by the basidiomycete fungus Moniliophthora perniciosa, a hemibiotrophic fungus that produces the necrosis and ethylene-inducting protein (MpNEP2) during infection; this protein can activate cysteine proteases and induce programmed cell death. Cysteine proteases can be modulated by cystatin. In this study, we overexpressed TcCYS4, a cocoa cystatin, in tobacco plants and evaluated the effect on MpNEP2 in model plants. Tccys4 cDNA was cloned into the pCAMBIA 1390 vector and inserted into the tobacco plants via Agrobacterium tumefaciens. Transgene expression was analyzed by reverse transcription-quantitative PCR and Western blot analysis. Transcript and protein levels in Tcccys4:tobacco lines were 8.9- and 1.5-fold higher than in wild-type plants (wt). Tcccys4:tobacco lines showed no change in growth compared to wt plants. CO2 net assimilation (A) increased in Tcccys4:tobacco lines compared to wt plants. Only one line showed statistically significant stomatal conductance (gs) and transpiration rate (E) changes. MpNEP2 was infiltered into the foliar mesophyll of Tcccys4:tobacco lines and wt plants, and necrotic lesions were attenuated in lines highly expressing Tccys4. Our results suggest that cocoa cystatin TcCYS4 affects MpNEP2 activity related to the progression of programmed cell death in tobacco plants. This may occur through the action of cystatin to inhibit cysteine proteases activated by MpNEP2 in plant tissues. Further studies are necessary to examine cystatin in the Theobroma cacao-M. perniciosa pathosystem.
在巴西，大部分可可豆生产集中在巴伊亚州南部。1989年，女巫扫帚病传入该地区，此后造成了严重的生产损失。这种疾病由担子菌纲真菌可可球二孢引起，它是一种半活体营养型真菌，在感染过程中会产生坏死和乙烯诱导蛋白（MpNEP2）；这种蛋白可激活半胱氨酸蛋白酶并诱导程序性细胞死亡。半胱氨酸蛋白酶可被胱抑素调节。在本研究中，我们在烟草植株中过表达了可可胱抑素TcCYS4，并评估其对模式植物中MpNEP2的影响。将Tccys4 cDNA克隆到pCAMBIA 1390载体中，并通过根癌农杆菌将其导入烟草植株。通过逆转录定量PCR和蛋白质印迹分析对转基因表达进行分析。Tcccys4烟草株系中的转录本和蛋白质水平分别比野生型植株（wt）高8.9倍和1.5倍。与wt植株相比，Tcccys4烟草株系的生长没有变化。与wt植株相比，Tcccys4烟草株系的二氧化碳净同化率（A）有所增加。只有一个株系的气孔导度（gs）和蒸腾速率（E）发生了统计学上的显著变化。将MpNEP2注入Tcccys4烟草株系和wt植株的叶肉中，在高表达Tccys4的株系中坏死病斑有所减轻。我们的结果表明，可可胱抑素TcCYS4影响与烟草植株程序性细胞死亡进程相关的MpNEP2活性。这可能是通过胱抑素抑制植物组织中被MpNEP2激活的半胱氨酸蛋白酶的作用来实现的。有必要进一步研究可可-可可球二孢病理系统中的胱抑素。
