Shivashankar S, Thimmareddy C, Roy Tapas K
Division of Plant Physiology and Biochemistry, Indian Institute of Horticultural Research, Bangalore 560 089, India.
Indian J Biochem Biophys. 2010 Aug;47(4):243-8.
Chilli fruit is highly susceptible to anthracnose infection at the stage of harvest maturity, due to which the fruit yield in the leading commercial variety Byadgi is severely affected. Field studies on screening of several varieties for resistance to anthracnose have shown that a variety of chilli AR-4/99K is resistant to anthracnose infection. In many crops, resistance to fungal attack has been correlated with PGIP activity in developing fruits based on which transgenic varieties have been developed with resistance to fungi. The present study was carried out to determine whether anthracnose resistance in AR-4/99K was due to the increased levels of PGIP alone and/ or due to differences, if any, in the properties of PGIP. Hence, a comparative study of the properties of polygalacturonase inhibitor protein (PGIP) isolated from fruits of anthracnose resistant chilli var AR-4/99K and a susceptible variety Byadgi was conducted with the objective of utilizing the information in genetic transformation studies. Both the PGIPs from anthracnose resistant and susceptible varieties of chilli exhibited similarities in the elution pattern on Sephadex gel, DEAE cellulose, PAGE and SDS-PAGE. The two PGIPs were active over a wide range of pH and temperature. Both PGIPs showed differential inhibitory activity against polygalacturonase (PG) secreted by Colletotrichum gleosporoides, C. capsici, C. lindemuthianum, Fusarium moniliforme and Sclerotium rolfsii. The inhibitory activity of PGIP from both resistant and susceptible varieties was the highest (82% and 76%, respectively) against the PG from Colletotrichum capsici, a pathogen causing anthracnose rot of chilli, while the activity was lower (1.27 to 12.3%) on the other fungal PGs. Although PGIP activity decreased with fruit maturation in both the varieties, the resistant variety maintained a higher activity at 45 days after flowering (DAF) as compared to the susceptible variety which helped it to overcome the infection by anthracnose as against the susceptible variety (Byadgi) in which PGIP activity was drastically reduced at maturity. The molecular mass of PGIP as determined by SDS-PAGE was found to be 37 kDa. N-terminal sequence analysis of the PGIP showed the first six amino acid residues from N-terminal end were Asp-Thr-His-Lys-Ser-Glu (DTHKSE), respectively. The similarities in properties of the two PGIPs support the earlier findings that resistance of AR-4/99K to anthracnose fungus is a result of its higher PGIP activity at maturity.
辣椒果实成熟采收阶段极易感染炭疽病，这使得主要商业品种拜德吉（Byadgi）的果实产量受到严重影响。对多个辣椒品种进行炭疽病抗性筛选的田间研究表明，辣椒品种AR - 4/99K对炭疽病感染具有抗性。在许多作物中，基于发育中果实的多聚半乳糖醛酸酶抑制蛋白（PGIP）活性与抗真菌侵袭能力相关，据此已培育出抗真菌的转基因品种。本研究旨在确定AR - 4/99K对炭疽病的抗性是否仅归因于PGIP水平的提高，以及PGIP特性是否存在差异（若有）。因此，对从抗炭疽病辣椒品种AR - 4/99K和感病品种拜德吉果实中分离得到的多聚半乳糖醛酸酶抑制蛋白（PGIP）特性进行了比较研究，目的是在遗传转化研究中利用这些信息。抗炭疽病和感病辣椒品种的PGIP在Sephadex凝胶、DEAE纤维素、聚丙烯酰胺凝胶电泳（PAGE）和十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳（SDS - PAGE）上的洗脱模式相似。两种PGIP在较宽的pH和温度范围内均有活性。两种PGIP对胶孢炭疽菌、辣椒炭疽菌、菜豆炭疽菌、串珠镰刀菌和齐整小核菌分泌的多聚半乳糖醛酸酶（PG）均表现出不同的抑制活性。抗感品种的PGIP对引起辣椒炭疽腐烂病的病原菌辣椒炭疽菌分泌的PG抑制活性最高（分别为82%和76%），而对其他真菌PG的活性较低（1.27%至12.3%）。尽管两个品种的PGIP活性均随果实成熟而降低，但与感病品种相比，抗感品种在开花后45天（DAF）时保持较高活性，这有助于其抵御炭疽病感染，而感病品种（拜德吉）在成熟时PGIP活性急剧下降。通过SDS - PAGE测定的PGIP分子量为37 kDa。PGIP的N端序列分析表明，从N端起的前六个氨基酸残基分别为天冬氨酸 - 苏氨酸 - 组氨酸 - 赖氨酸 - 丝氨酸 - 谷氨酸（DTHKSE）。两种PGIP特性的相似性支持了早期的研究结果，即AR - 4/99K对炭疽病菌的抗性是其成熟时较高的PGIP活性的结果。
