SummaryAgrobacterium exports at least five virulence proteins (VirE2, VirE3, VirF, VirD2, VirD5) into host cells and hijacks some host plant factors to facilitate its transformation process.Random DNA binding selection assays (RDSAs), electrophoretic mobility shift assays (EMSAs) and yeast one-hybrid systems were used to identify protein-bound DNA elements. Bimolecular fluorescence complementation, glutathione S-transferase pull-down and yeast two-hybrid assays were used to detect protein interactions. Protoplast transformation, coprecipitation, competitive binding and cell-free degradation assays were used to analyze the relationships among proteins.We found that Agrobacterium VirD5 exhibits transcriptional activation activity in yeast, is located in the plant cell nucleus, and forms homodimers. A specific VirD5-bound DNA element designated D5RE (VirD5 response element) was identified. VirD5 interacted directly with Arabidopsis VirE2 Interacting Protein 1 (AtVIP1). However, the ternary complex of VirD5-AtVIP1-VirE2 could be detected, whereas that of VirD5-AtVIP1-VBF (AtVIP1 Binding F-box protein) could not. We demonstrated that VirD5 competes with VBF for binding to AtVIP1 and stabilizes AtVIP1 and VirE2 in the cell-free degradation system.Our results indicated that VirD5 may act as both a transcriptional activator-like effector to regulate host gene expression and a protector preventing the coat proteins of the T-complex from being quickly degraded by the host's ubiquitin proteasome system (UPS).

Efficient photosynthesis requires a balance of ATP and NADPH production/consumption in chloroplasts, and the exportation of reducing equivalents from chloroplasts is important for balancing stromal ATP/NADPH ratio. Here, we showed that the overexpression of purple acid phosphatase 2 on the outer membranes of chloroplasts and mitochondria can streamline the production and consumption of reducing equivalents in these two organelles, respectively. A higher capacity of consumption of reducing equivalents in mitochondria can indirectly help chloroplasts to balance the ATP/NADPH ratio in stroma and recycle NADP+, the electron acceptors of the linear electron flow (LEF). A higher rate of ATP and NADPH production from the LEF, a higher capacity of carbon fixation by the Calvin–Benson–Bassham (CBB) cycle and a greater consumption of NADH in mitochondria enhance photosynthesis in the chloroplasts, ATP production in the mitochondria and sucrose synthesis in the cytosol and eventually boost plant growth and seed yields in the overexpression lines.

During Agrobacterium (Agrobacterium tumefaciens) infection, the translocated virulence proteins (VirD2, VirE2, VirE3, VirF and VirD5) play crucial roles. It is thought that, through protein-protein interactions, Agrobacterium uses and abuses host plant factors and systems to facilitate its infection. Although some molecular functions have been revealed, the roles of VirD5 still need to be further elucidated. Here, plant transformation and tumorigenesis mediated by genetically modified Agrobacterium strains were performed to examine VirD5 roles. In addition, protein-protein interaction-associated molecular and biochemistry technologies were used to reveal and elucidate VirD5 interaction with Arabidopsis VirE2 interacting protein 2 (VIP2). Our results showed that deleting virD5 from Agrobacterium reduced its tumor formation ability and stable transformation efficiency but did not affect the transient transformation efficiency. We also found that VirD5 can interact with Arabidopsis VIP2. Further experiments demonstrated that VirD5 can affect VIP2 binding to cap-binding proteins (CBP20 and CBP80). The tumorigenesis efficiency for cbp80 mutant was not significantly changed, but that for cbp20, cbp20cbp80 mutants were significantly increased. This work demonstrates experimentally that VirD5 is required for efficient Agrobacterium infection and may promote this process by competitive interaction with Arabidopsis VIP2. CBP20 is involved in the Agrobacterium infection process and its effect can be synergistically enhanced by CBP80.

Efficient photosynthesis requires a balance of ATP and NADPH production/consumption in chloroplasts as the linear electron flow generates a higher NADPH/ATP ratio than that is consumed by the Calvin-Benson-Bassham cycle. Recent works suggested that ATP importation into mature chloroplasts of Arabidopsis thaliana is negligible, and therefore the exportation of reducing equivalents from chloroplasts is important for balancing stromal ATP/NADPH ratio. Here we showed that the overexpression of purple acid phosphatase 2 on the outer membranes of chloroplasts and mitochondria can streamline the production and consumption of reducing equivalents in these two organelles, respectively. A higher capacity of consumption of reducing equivalents in mitochondria can indirectly help chloroplasts to balance the ATP/NADPH ratio in stroma and recycle NADP+, the electron acceptors of the linear electron flow. A higher rate of ATP and NADPH production from the linear electron flow, a higher capacity of carbon fixation by the Calvin-Benson-Bassham cycle and a greater consumption of NADH in mitochondria, enhance photosynthesis in the chloroplasts, ATP production in the mitochondria, sucrose synthesis in the cytosol, and eventually boosting plant growth and seed yields in the overexpression lines.One-Sentence SummaryThis study demonstrates the importance of chloroplast-mitochondria cooperation in redox balance and illustrates that an optimized function of mitochondria can enhance the efficiency of photosynthesis.

Large and complex maize BIBAC inserts, even with a length of about 164 kb and repeat sequences of 88.1%, were transferred into rice. The BIBAC vector has been established to clone large DNA fragments and directly transfer them into plants. Previously, we have constructed a maize B73 BIBAC library and demonstrated that the BIBAC clones were stable in Agrobacterium. In this study, we demonstrated that the maize BIBAC clones could be used for rice genetic transformation through Agrobacterium-mediated method, although the average transformation efficiency for the BIBAC clones (0.86%) is much lower than that for generally used binary vectors containing small DNA fragments (15.24%). The 164-kb B73 genomic DNA insert of the BIBAC clone B2-6 containing five maize gene models and 88.1% of repetitive sequences was transferred into rice. In 18.75% (3/16) of the T1, 13.79% (4/29) of the T2, and 5.26% (1/19) of the T3 generation transgenic rice plants positive for the GUS and HYG marker genes, all the five maize genes can be detected. To our knowledge, this is the largest and highest content of repeat sequence-containing DNA fragment that was successfully transferred into plants. Gene expression analysis (RT-PCR) showed that the expression of three out of five genes could be detected in the leaves of the transgenic rice plants. Our study showed a potential to massively use maize genome resource for rice breeding by mass transformation of rice with large maize genomic DNA fragment BIBAC clones.