Interaction Study And Function Verification Of Virulence Proteins And Host Factors In Agrobacterium-mediated Transformation

Agrobacterium mediated transformation is one of the widely used method to cratetransgenic plants. Agrobacterium genetically transforms plants by transferring virulenceprotein factors and T-DNA to host cells and integrating T-DNA into host genome. Thisprocess requires both proteins coming from both Agrobacterium and plants. TransferredT-DNA may form complexes with virulence proteins and host proteins. This complex isimportant for protecting T-DNA from hydrolytic degradation by nuclease, the transportand chromatin targeting and the proteolysis of the coated proteins of T-DNA complex.Agrobacterium factors which are involved in transformation have been studied pettyclearly, but not the plant factors. There still have many protein factors to screen, evenfunctions of most of the known proteins which affect Agrobacterium mediatedtransformation are not clear. This article will talk about functions of several hostproteins which affect transformation process and a new model for the role of VirE2-VIP1interactions in plant transformation process was proposed, the following results wereobtained:1. VIP1(VirE2interacting protein1), an Arabidopsis bZIP protein, has beensuggested to mediate transformation through interaction with and targeting of VirE2tonuclei. Susceptibilities of Arabidopsis vip1mutant and VIP1(VIP1、VIP1S79AandVIP1S79D) overexpressing plants to transformation by numerous Agrobacterium strainswere examined use root segment assay method. In no instance could we detect alteredtransformation susceptibility.2. The subcellular localization of Venus-tagged VirE2or Venus-tagged VIP1wasalso examined use confocal microscopy, in the presence or absence of the other untaggedprotein, in roots of transgenic Arabidopsis, agroinfiltrated Nicotiana benthamiana leaves,mesophyll protoplasts of Arabidopsis leaves and tobacco BY-2protoplasts. We foundthat VIP1-Venus localized in both the cytoplasm and the nucleus of these plant cell systems, regardless of whether there was co-expressed VirE2or not. VirE2localized tothe cytoplasm of these plant cell systems, regardless of whether there was co-expressedVIP1or not. The Ser79of VIP1does not affect the subcellular localization of VIP1.The conclusion is that VIP1is not important VirE2subcellular localization. Comparedto their wild type plants, vip1-1mutant and VIP1(VIP1S79A，VIP1S79D) overexpressingplants did not show any difference in Agrobacterium-mediated transformation.3. With bimolecular fluorescence complementation (BiFC) and confocal microscopy,it was found that VIP1-nVenus interacted with VirE2-cCFP in the cytoplasm ofArabidopsis leaf protoplasts and in tobacco BY-2protoplasts. Multicolor BiFCindicated exclusive cytoplasmic localization of both VirE2-VIP1and VirE2-VirE2complexes.4. Considering the importance of VirE2in Agrobacterium-mediated transformation,the predominant cytoplasmic localization of VirE2, the cytoplasmic localization ofVirE2-VIP1complexes, and the role of VIP1as a positive regulator of plant defenseresponses, a new model for the role of VirE2in Agrobacterium-mediated transformationwas proposed. In this model, nuclear targeting of T-strands is manly effected by VirD2,although VirE2may play a “structural” role in “winding or unwinding” structures inT-strands, thus facilitating nuclear entry of T-strand. In plant cells, in addition to the“structural” role, VirE2may interact with VIP1and prevents at least part of the smallendogenous VIP1pool from going to nucleus and serves as a transcriptional modulator,so that VIP1could not activate plant defense genes effectively, thus facilitatingtransformation.5. Considering the importance of virulence proteins in Agrobacterium mediatedtransformation, changed transformed transformation efficiency of some actin mutantArabidopsis and myosin mutant Arabidopsis, and the relationship between myosin andactin, transgenic Col-0、myosin VIII1/A、VIII2、VIII1/2/A、VIII1/2/A/B、XI-H andXI1/K plants which express VirD2-Venus、Venus-VirE2and Venus-VirF separately werecreated. Confocal microscopy results indicate that the subcellular localization of thethree protein do not change, it means the decreased transformation in these myosinmutants was not resulted from affecting VirD2and VirF’s subcellular localization. InCol-0、VIII1/A、VIII2、VIII and VIII1/2/A/B mutants, VirE2localizes in cytoplasm, itindicated the changed transformation efficiency was not caused by changing VirE2’s subcellular localization. VirE2localized to cytoplasm and nucleus in XIH mutant, andto cytoplasm in XI1/K mutant but tend to form aggregates more easily. It is stillunknown how the nuclear localization or easier to form aggregates affect XIH andXI1/K’s transformation susceptibility separately.