Exploring the biochemical function of RolB and RolC proteins

Many studies have indicated that rol genes affect plant in lots of aspects. In our study, we found that rolB and rolC are dominant factors among all four rol genes in

hairy root initiation, growth promotion, and growth activity maintenance. We hypothesized that RolB and RolC regulate plant physiology via altering plant signaling or transcription process. To understand which pathway(s) may be regulated, we conducted yeast two-hybrid screening to find out the protein(s) associated with RolB or RolC protein.

To perform yeast two-hybrid assay, we constructed chimeric proteins of the respective Rol protein fused with GAL4-DNA binding domain, and we constructed the cDNA library from fast-growing and high nicotine accumulating hairy root clone 9. By screening, we discovered that RolB may interact with three proteins, including a basic leucine zipper domain (bZIP domain)-containing protein NtbZIP, the phosphate-induced gene encoding protein PHI-2, and ORF13a (Sung-Hui Yi, 2014).

The first two proteins are found in tobacco, and the ORF13a is encoded by A.

rhizogenes T-DNA. Notably, all of these three proteins are DNA-associated proteins containing conserved serine/threonine phosphorylation motif (Hansen et al. 1994;

Jakoby et al. 2002; Sano and Nagata 2002), and the bZIP containing protein and the PHI-2 protein have been demonstrated as an abscisic acid (ABA) responsive proteins.

These hinted RolB may regulate ABA signaling by transcriptional steps.

By sequence analysis, RolB does not contain typical nucleus localization sequence. However, the previous study showed RolB is translocated into nucleus while physical interaction with 14-3-3 proteins (Moriuchi et al. 2004). We expressed eYFP-RolB fusion protein in both Arabidopsis and Nicotiana tabacum protoplasts, and the eYFP signals were found in nucleus (Ta-Chung Lin, unpublished). Even though RolB does not contain typical nucleus translocation signal peptide, Moriuchi and colleagues discovered that RolB can interact with Arabidopsis thaliana 14-3-3

proteins, which resulting in nucleus translocation of RolB-14-3-3 protein complex (2004). In our yeast two-hybrid assay system, we did not found any 14-3-3 proteins that can interact with RolB; however, tobacco 14-3-3
I can interact with RolB by

bimolecular fluorescence complementation (BiFC) assay, and the fluorescent signal appeared clearly in nucleus (Ta-chung Lin, unpublished). Combining these data, we hypothesized that RolB suppresses ABA signaling in transcriptional process to result in an “auxin-like” effect, which increases cell proliferation and differentiation to promote rooting and growing.

In addition to PHI-2 and NtbZIP, ORF13a may also involved in RolB signaling.

The early research reported that rolB gene and orf13, which also contains orf13a in their study, can act synergistically to promote rooting more efficiently than rolB alone (Aoki and Syõno 1999). Combined the former study, our results suggested RolB and ORF13a may work together to induce rooting. However, the detail mechanism should be further elucidated.

All of NtbZIP, PHI-2, and ORF13a do not have typical tyrosine phosphorylation motif; instead, they have conserved serine/threonine phosphorylation motifs. If these proteins share the similar regulatory mechanism with typical ABA-responsive transcriptional factors, the phosphorylation of these proteins is important to activate down-stream ABA-related transcription processes (reviewed by Fujita et al. 2013).

However, Filippini and coworkers found the protein extract from rolB-expressing E.

coli showed significant higher phosphatase activity than non-transformed E. coli by using universal phosphatase substrate para-nitrophenylphosphatase (pNPP). The phosphatase activity can be reduced by adding tyrosine phosphatase inhibitors, but neither serine phosphatase inhibitors nor threonine phosphatase inhibitors was able to

repress the activity. Moreover, they performed RolB phosphatase activity assay with tyrosine-, serine-, threonine-phosphorylated peptides, and RolB can only release free phosphate group from tyrosine-phosphorylated peptide. They concluded that RolB is a tyrosine phosphatase (1996). To confirm whether RolB has phosphatase activity, we expressed a N-terminal GST fused RolB (GST-RolB) in E. coli. Neither crude extract nor affinity-purified GST-RolB proteins showed phosphatase activity on the universal phosphatase substrate pNPP in our system. We then considered the GST tag may affect the enzymatic activity. To prove this, we construct C-terminal GST fused RolB proteins as well as tag-free RolB proteins to assay the phosphatase activity;

nevertheless, none of the crude extracts exhibited phosphatase activity as well. To check with more confidences, we will express RolB in tobacco protoplast and perform the phosphatase assay again by the plant-expressing RolB protein.

In addition to direct de-phosphorylation, RolB might repress the activities of RolB-interacting proteins, including PHI-2, NtbZIP, and ORF13a, via indirect de-phosphorylation or degradation. To prove this, we will mimic the de-phosphorylation and the de-phosphorylation of PHI-2 and NtbZIP by replacing the predicted Ser/Thr into alanine and glutamic acid, respectively. Then we will assay if the point mutation change the result of protein-protein interaction. It will reveal whether the interaction is phosphorylation dependent or not.

On the other hand, RolC fused with Gal4-DNA binding domain (RolC::DNA-BD) exhibited a strong auto-activation in yeast two-hybrid system. The selection markers include 2 basic nutrients, histidine and adenine, biosynthesis genes HIS3 and ADE2, an antibiotic Aureobasidin A (AbA) resistant gene AUR1-C, which encodes inositol phosphorylceramide synthase, and a blue/white selection reporter gene

MEL1, which encoded alpha-galactosidase (Figure 4-2). The auto-activation activity of RolC indicated that RolC might be a transcriptional factor (Figure 4-2). However, Estruch and coworkers discovered that RolC appeared in cytosolic fraction (1991).

Also, we expressed eYFP-RolC in both Arabidopsis and Nicotiana tabacum protoplasts, and the eYFP signals were found in the cytoplasms (Ta-Chung Lin and Ke-Jin Lin, unpublished). These results lowered the possibility that RolC is a transcriptional factor.

Figure 4-2 RolC has transcriptional activity in yeast. To perform yeast two-hybrid assay, we fused respective rol genes with Gal4-DNA binding domain, and we found the rolC itself can strongly activate the reporter genes without Gal4 activation domain.

In order to find the RolC interacting proteins, we have to eliminate the auto-activation activity. We have tried to grow the yeast harboring RolC::DNA-BD with histidine and adenine deficient medium and 1.5-fold higher concentration than standard usage of Aureobasidin A; however, it can grow normally. We therefore fuse the RolC with Gal4-DNA activation domain (RolC::DNA-AD) and try to do a screening with the prey fused with Gal4-DNA binding domain. Nevertheless,

RolC::DNA-AD causes yeast lethal. Moreover, RolC is a small protein with approximate 20 kD in molecular weight; therefore, we do not propose to use partial RolC to do the yeast two-hybrid screening. To overcome these problem, we are performing an error-prone PCR to generate a serial mutated rolC to find the mutants that cannot auto-activate the reporter. To date, only proline mutation, stop codon incorporation, and frame-shift by nucleotide insertion or deletion mutants have been identified. We hope we can got more diverse kinds of mutants. Then, we will apply some of mutated RolC to yeast two-hybrid screening to find out the putative protein candidates, and we will do co-immunoprecipitation assay and bimolecular complementation assay to check if the candidates can interact with native RolC.

3. The mechanism of secondary metabolite accumulation in hairy roots