A method for ROP protein activity detection in vivo and its application

Abstract: 【Objective】This work aimed to establish a method for detecting ROP protein activity in plants and to provide some technological support for further studies on the function of ROP proteins. 【Methods】The working principle of the method was mainly based on the fact that RIC, as an effector of the ROP protein, specifically binds to the active form of ROP proteins. Through the luciferase complementation assay, with the help of the Agrobacterium-mediated tobacco (Nicotiana benthamiana) transient expression system, the plant live imager and microplate reader were used to qualitatively or quantitatively detect the fluorescence intensity, to achieve the detection of ROP protein activity in vivo.【Results】In this study, the AtRIC1 was used as the test gene and AtROP1 was used as an example to detect its protein activity. By this method, a strong fluorescent signal was successfully detected in the co-injection region of AtROP1 and AtRIC1. The test results indicated that AtROP1 had an interaction with AtRIC1 and this method could detect the activity of AtROP1 protein. In addition, the AtROP1 protein was subjected to a targeted mutation assay, in which glutamine (Gln or Q) at site 64 was mutated to leucine (Leu or L) to create a persistently activated ROP1 (AtROP1-CA) vector, and aspartic acid (Asp or D) at site 121 was mutated to alanine (Ala or A) to create a persistently inactivated ROP1 (AtROP1-DN) vec-tor. The two vectors (AtROP1-CA-nLUC and AtROP1-DN-nLUC) were then successfully constructed and transformed into Agrobacterium GV3101. The changes in the activity of AtROP1 protein after the targeted mutation were tested again by this method. The qualitative and quantitative results showed that the luciferase fluorescence intensity and activity values were significantly enhanced in the AtROP1-CA treated group (6 605.0±209.2) compared with the control AtROP1 proteins (4 395.0±103.1), which indicated that the AtROP1-CA protein showed higher activity. In contrast, the luciferase fluorescence intensity and activity values of AtROP1-DN protein (1 134.0±39.83) were significantly lower compared with the control AtROP1 protein (4 366.0±286.7), indicating that AtROP1-DN protein had only very low activity. These results suggested that AtRIC1 could be used as a test protein to detect changes in the intensity of AtROP1 protein activity. Besides this, the reliability of the method was further tested by introducing a third protein (AtGAP1, AtGDI1 and AtGEF1 proteins as examples). Initially, the AtGAP1, AtGDI1 and AtGEF1 genes were successfully constructed into the p1300-35S-GFP vector and they were transformed into Agrobacterium GV3101. The upstream regulators (AtGAP1- GFP and AtGDI1- GFP) that inhibited AtROP1 activity were again verified by this method to certify whether they also inhibit AtROP1 protein activity. The test results showed that compared with the control group and the treatment group with the addition of GFP in the empty load (5 035.0±121.5), the luciferase fluorescence intensity and activity values of the AtROP1 protein in the AtGAP1-GFP treatment group (1 473.0±146.6) both showed a significant decrease, and both GFP and AtGAP1-GFP proteins were indeed expressed in this system, and that the expression of AtGAP1-GFP indeed reduced the activity of AtROP1 protein. Similarly, the AtGDI1-GFP treated group (1 621.0±85.41) showed a significant decrease in luciferase fluorescence intensity and activity values of the AtROP1 protein compared with the control group and the treatment group with the addition of GFP in the empty load (4 798.0±145.0), while it was also detected that GFP and AtGAP1-GFP proteins were indeed expressed in this system, and that the expression of AtGDI1-GFP also indeed reduced the activity of the AtROP1 protein. AtGEF1, a regulator upstream of AtROP1 activity, was able to activate the activity of the ROP protein. To confirm the applicability of this method, an AtGEF1-GFP vector was successfully constructed. The test results revealed that the luciferase fluorescence intensity and activity values of the AtROP1 protein were significantly enhanced in the AtGEF1-GFP-treated group (7 338.00±83.83) compared with the control group and the treatment group with the addition of GFP in the empty load (4 704.0±133.6), and it was also detected that GFP and AtGEF1-GFP proteins were indeed expressed in this system, and the expression of AtGEF1-GFP did increase the activity of the AtROP1 protein. The above results suggested that this method could also detect the effect of other proteins on AtROP1 protein activity through the AtRIC1 protein. This method also would solve the problem of the previous single pull-down assay to detect the change of ROP protein activity, and add a new method to detect ROP protein activity in vivo.【Conclusion】At the moment, the typical method for the detection of ROP protein activity was to pull-down the active ROP protein from the extracted total plant protein (or purified protein) using the MBP-RIC1 protein purified in advance. This method could be used to determine changes in the activity of the ROP protein of the target sample. Compared with the traditional pull-down method, this method has the characteristics of high sensitivity, visualization, quantification and simple operation. It also would be suitable for detecting the effect of other proteins on the activity of ROP protein, so this method seems to be a reliable new method for detecting the activity of ROP protein in vivo.