Nox Complex signal and MAPK cascade pathway are cross-linked and essential for pathogenicity and conidiation of mycoparasite Coniothyrium minitans

The NADPH oxidase complex of a sclerotial mycoparasite Coniothyrium minitans, an important biocontrol agent against crop diseases caused by Sclerotinia sclerotiorum, was identified and its functions involved in conidiation and mycoparasitism were studied. Gene knock-out and complementary experiments indicated that CmNox1, but not CmNox2, is necessary for conidiation and parasitism, and its expression could be significantly induced by its host fungus. CmNox1 is regulated by CmRac1-CmNoxR and interacts with CmSlt2, a homolog of Saccharomyces cerevisiae Slt2 encoding cell wall integrity-related MAP kinase. In ΔCmNox1, CmSlt2-GFP fusion protein lost the ability to localize to the cell nucleus accurately. The defect of conidiation in ΔCmRac1 could be partially restored by over-expressing CmSlt2, indicating that CmSlt2 was a downstream regulatory factor of CmNox1 and was involved in conidiation and parasitism. The expressions of mycoparasitism-related genes CmPks1, Cmg1 and CH1 were suppressed in the knock-out mutants of the genes in CmNox1-CmSlt2 signal pathway when cultivated either on PDA. Therefore, our study infers that CmRac1-CmNoxR regulates CmNox1-CmSlt2 pathway in regulating conidiation and pathogenicity of C. minitans.


Results
NADPH oxidases (Nox1/Nox2) in C. minitans. Two NADPH oxidase genes, CmNox1 and CmNox2, were isolated from C. minitans. The deduced amino acid sequence of CmNox1 (GenBank Accession No: KJ596434) shows high similarity to Nox1 homologs from other filamentous fungi, including Alternaria alternate (AaNox1, BAK52527.1, 89% identity), Curvularia lunata (ClNOX1, AHC53982.1, 89% identity), and Pyrenophora tritici-repentis (PrNOX1, XP_001935118.1, 88% identity). CmNox2 (GenBank Accession No: KJ596435) is highly similar to Nox2 homologs from other filamentous fungi. The multiple alignment analysis showed that both CmNox1 and CmNox2 contained NOX family signature regions. Phylogenetic analysis of NADPH oxidases in several fungi placed CmNox1 and CmNox2 homologs into two different clades based on the amino acid sequences (Supplementary Figure S1). CmNox1 is essential for ROS production and conidiation. To study the function of CmNox1 in C. minitans, a replacement vector p3300neoCmNox1 (see Supplementary Figure S2a) was constructed and transformed into strain ZS-1 to disrupt CmNox1. Twenty transformants were obtained, and three of which, ΔCmNox1-1, ΔCmNox1-6, and ΔCmNox1-107 were selected randomly as candidates for further analyses. Furthermore, a complement vector pNox1 was transformed into mutant ΔCmNox1-6. The deletion and complementary events were confirmed by RT-PCR (see Supplementary Figure S2b) and Southern blot analysis (see Supplementary Figure S2c). Using the same methods, a replacement vector p3300neoCmNox2 was constructed (see Supplementary Figure S2a) and transformed into strain ZS-1 to disrupt CmNox2. Three deletion mutants ΔCmNox2-20, ΔCmNox2-323 and ΔCmNox2-347 were also confirmed by Southern blot analysis.
Colony staining with NBT solution showed that superoxide production was decreased significantly in ΔCmNox1-6, compared to ΔCmNox2-323 and the wild-type strain ZS-1 (Fig. 1a). The conidiation of the wild-type strain ZS-1, CmNox1 deletion mutants, CmNox1 complemented mutants, and CmNox2 deletion mutants were determined after incubating for 15 days on PDA (Fig. 1b). CmNox1 deletion mutants completely lost the ability to produce conidia (Table 1). In contrast, the wild-type strain ZS-1, CmNox1 complemented mutants and CmNox2 deletion mutants were normal in conidiation under the same condition (Table 1). Unlike strain ZS-1 and ΔCmNox2-323, which could form matured pycnidia and conidia, ΔCmNox1-6 could only form a few pycnidial primordia that could not further develop to mature pycnidia, and no conidium was produced (Fig. 1b). These data indicated that CmNox1, but not CmNox2, played significant roles in conidiation and production of superoxide. The experiments also suggested CmNox1 and CmNox2 are not essential for hyphal growth of C. minitans on PDA.
CmNox1 is essential to parasitize sclerotia of S. sclerotiorum. To determine whether CmNox1 is related to sclerotial parasitizing, the parasitic ability of CmNox1 mutants and the other strains to sclerotia of S. sclerotiorum were examined. The hyphae of ΔCmNox1-6 was inoculated on to sclerotia and incubated for 30 days at 20 °C, and no pycnidia and conidia were observed on either the surface or interior of the sclerotia (Fig. 2a, Table 1). The inoculated sclerotia were surface sterilized and then incubated on PDA containing 50 μg/ml hygromycin, and no C. minitans colony emerged from the sclerotia, suggesting that ΔCmNox1-6 did not invade the inner of sclerotia (Fig. 2b, Table 1). Meanwhile, CmNox1-C8, ΔCmNox2-323 and the wild-type strain ZS-1 could degrade sclerotia and produce mature pycnidia there. The results demonstrated CmNox1 played a significant role in sclerotial mycoparsitism.
Expression of CmNox1 is highly induced by S. sclerotiorum. When dual cultured with S. sclerotiorum, the expression of CmNox1 was highly induced. Compared to growing on PDA, the expression of CmNox1 peaked at 12 hpi, and the high expression was maintained till 24 hpi, and then sharply declined to undetectable level at 36 hpi (Fig. 3a). This phenomenon suggested that CmNox1 played an important role in parasitism. Expression of CmNox2 also was up-regulated slightly by S. sclerotiorum at 12 hpi and 24 hpi, but compared to culture on PDA, the induction was much lower (Fig. 3b). The results showed that S. sclerotiorum could induce the expression of CmNox1 and CmNox2, with stronger induction to CmNox1. CmRac1 interacts with CmNoxR together to regulate CmNox1 and to control conidiation in C. minitans. It is reported that Bem1 and Cdc24 are components of the NADPH oxidase complex in filamentous fungi, and NoxR interacting with RacA in vitro together regulates ROS production and control hyphal branching and patterning in E. festucae 27,29 . Since CmNox1 is involved in the production ROS in C. minitans, we identified C. minitans homologs of NoxR, GTPase Rac1 and Bem1, named CmNoxR (GenBank Accession No: KJ596436), CmRac1 (GenBank Accession No: KJ596436) and CmBem1, respectively. The interaction between CmRac1 and CmNoxR, CmBem1 and CmNoxR were confirmed by Yeast two-hybrid (Fig. 3c,d).
To investigate the role of CmNoxR, CmBem1 and CmRac1 in C. minitans, replacement vector for CmNoxR (p3300neoCmNoxR), CmRac1 (p3300neoCmRac1), and CmBem1 (p3300neoCmBem1) were constructed and transformed into ZS-1. The CmNoxR deletion mutants (ΔCmNoxR-5 and ΔCmNoxR-107), CmBem1 deletion mutant (ΔCmBem1-344) and CmRac1 deletion mutant (ΔCmRac1-79) were obtained and confirmed by RT-PCR. In contrast to the wild-type strain ZS-1, the CmNoxR deletion mutants and the CmRac1 deletion mutant lost the ability to produce conidia and to parasitize S. sclerotiorum. In addition to the conidiation and parasitizing phenotype, growth rate also was reduced significantly in the CmRac1 deletion mutant. The growth rate of ΔCmRac1-79 was less than 0.6 mm/day on average on PDA, while it was about 3mm/day for strain ZS-1 under the same conditions. However, there was no significant difference in the growth rate between CmNoxR deletion mutants and ZS-1, and the deletion mutant displayed a phenotype similar to CmNox1 deletion mutant (Fig. 3e, Table 1). ΔCmBem1-344 shared the same phenotype as strain ZS-1. It suggests that CmRac1and CmNoxR are required to activate CmNox1 to adjust conidiation and parasitism, while CmBem1 is not involved.
CmSlt2 is CmNox1 effector required for conidiation and parasitism. To understand the mechanism of CmNox1-mediated conidiation and parasitism in C. minitans, we further investigated the functional relationship between CmNox1 and CmSlt2. We reported previously that CmSlt2 (cell wall integrity-related MAP kinase) is essential for conidiation of C. minitans. ΔCmSlt2 lost the ability to produce pycnidia and conidia, and had dramatically reduced sclerotial mycoparasitism 13 . The phenotype of ΔCmSlt2 was similar to that of ΔCmNox1, thus CmSlt2 and CmNox1 might have somewhat interaction directly or indirectly. To identify whether CmSlt2 interact with CmNox1, full length gene of CmSlt2 and CmNox1 were cloned into yeast two-hybrid vector pGADT7 and pGBDT7 to test the possible interaction, and the results showed that CmSlt2 could interact with CmNox1 (Fig. 4a). Furthermore, we generated fusions of CmSlt2 and CmNox1 with N-and C-terminal domains of YFP and assessed their interaction in tobacco (Nicotiana benthamiana) using bimolecular fluorescence complementation (BiFC) (Fig. 4c). To confirm this interaction in C. minitans, a transformant with TrpC-CmNox1-Flag and TrpC-CmSlt2-GFP and a transformant with TrpC-CmSlt2-GFP and Vector-Flag in ZS-1 were constructed. In Western blot analysis with total protein, the anti-Flag and anti-GFP antibodies detected a 65-kDa and an 80-kDa band, respectively. In proteins eluted from anti-Flag agarose, the 80-kDa CmSlt2-GFP band was detected with an anti-GFP antibody in transformant with CmSlt2 and CmNox1, but not in transformant with CmSlt2 only (Fig. 4b). The results showed that CmSlt2 and CmNox1 interacted directly in C. minitans.
We expressed the CmSlt2-GFP fusion in the wild-type strain ZS-1 and ΔCmNox1, and found that over-expressing CmSlt2-GFP could not rescue the deficiency of conidiation and parasitism in ΔCmNox1 (Fig. 5a,b). In the wild-type strain ZS-1, CmSlt2 was located in the cytoplasm during the hyphal growth period (3-day-old hyphae), and was located in the nucleus in 40% of the hyphae at the later stage of conidiation (5-day-old hyphae) observed under a fluorescence microscope; While in ΔCmNox1, no fluorescence signal of CmSlt2-GFP was observed in the nucleus at any stages (Fig. 5c). The results suggested that CmSlt2 was a downstream regulatory factor of CmNox1 and was involved in conidiation and parasitism.

Strains
Growth rate a (mm/d) Condiation b (x10 7 condia/plate) Rot index c (%) Over expressing CmSlt2 in ΔCmRac1 could partially restore conidiation and parasitism. Since CmRac1 interacts with CmNoxR together in regulating CmNox1, and CmNox1 mediates the localization of CmSlt2 in nucleus, we suspected that CmRac1 is likely to regulate the expression of CmSlt2. qRT-PCR analysis showed that the expression of CmSlt2 was obviously decreased in ΔCmRac1 by 0.5 fold, compared to the wild-type strain (Fig. 6a). We fused CmSlt2 with a trpC promoter from Aspergillus and transformed into ΔCmRac1, and found that over-expressing of CmSlt2 could partially restore conidiation and parasitic ability of ΔCmRac1 (Fig. 6b, and Table 1), in spite of no significant improvement of the hyphal growth.
The expression of CmPks1 was suppressed in CmNox1 signal pathway mutants. C. minitans produces dark pigment and black pycnidia during the late stage of growth. However, all ΔCmRac1, ΔCmNoxR, ΔCmNox1, and ΔCmSlt2 produce little dark pigment, and colonies were whitish. It is likely that the expression of CmPks1, a melanin biosynthesis associated polyketide synthase-encoding gene, was suppressed in those mutants. The transcript profile of CmPks1 was monitored by qRT-PCR, and the result showed that the expression of CmPks1 peaked at 96 hpi in the wild type ZS-1 and was significantly suppressed in ΔCmNoxR, ΔCmRac1, ΔCmNox1 and ΔCmSlt2, whereas there was no obvious difference in the transcript level between ΔCmNox2 and ZS-1 (Fig. 7a). Further experiments proved that the expression of CmPks1 in ZS-1 was enhanced by 300-400 fold when dual cultured with S. sclerotiorum than cultured on PDA alone at 24 hpi and 36 hpi (Fig. 7b), while it could not be induced in ΔCmRac1 and ΔCmNox1 (Fig. 7c). These results indicated that the expression of CmPks1 could The CmNox1 signal pathway is involved in regulating cell wall degrading enzyme genes. The parasitic ability of all mutants in CmNox1 signal pathways decreased obviously and it is also reported that the cell wall degrading enzyme Cmg1 (β -1,3 glucanase-encoding gene) and CH1 (chitinase gene) play important roles in the process of parasitizing sclerotia of S. sclerotiorum 9 . Our results of qRT-PCR demonstrated that the transcript levels of both Cmg1 and CH1 in ZS-1 were enhanced significantly at 24 hpi and 36 hpi when dual cultured with S. sclerotiorum (Fig. 7e,h), while were suppressed in ΔCmNoxR, ΔCmRac1, ΔCmNox1 or ΔCmSlt2 when incubated on PDA compared to that in ZS-1 (Fig. 7d,f,g,i). The results indicated that Cmg1 and CH1 were involved in parasitism and could be regulated by the CmNox1 signal pathway.

Discussion
The mycoparasitism system of C. minitans/S. sclerotiorum is unique and important to probe fungi and fungi interaction. In this study, the role of NADPH oxidases complex of C. minitans on growth, conidiation and mycoparasitism were studied. We found that CmNox1, but not CmNox2, played essential roles on conidiation and mycoparasitism. Furthermore, we found that CmRac1 could interact with CmNoxR, and CmNox1 could interact with CmSlt2. Expression of CmSlt2 in ΔCmRac1 could partially restore the conidiation and parasitism. These  21 . Nox1 is required for penetration, hyphal elongation, and Nox2 is required for assembly of a toroidal F-actin network during the penetration peg formation 22 ; In S. sclerotiorum, Nox1 is required for virulence, oxalic acid production and sclerotial development, while, Nox2 might be involved in sclerotial development 24 . In P. anserine, PaNox1 mutants are impaired in the differentiation of fruiting bodies from their progenitor cells, and deletion of the PaNox2 specifically blocked ascospore germination 33,54 . New Nox proteins were also identified from P. anserine, a Nox isoform Nox3 was found to play a minor role 36 . In N. crassa, Nox1 is required for female sterility, and involved in asexual development and hyphal growth, while Nox2 might be involved in ascospore germination 34 . In E. festucae, both ΔNoxA and ΔNoxB mutants could produce conidia, while NoxA, but not NoxB, is essential for hyphal polarized growth and hyphal funsion, NoxB did not affect conidiation 29 . In Sordaria macrospora, Nox1 is required for fruiting body formation, normal hyphal growth, and hyphal fusion, while Nox2 is involved in strict melanin-dependent ascospore germination 55 . In C. purpurea, CpNox1 is essential for infection, but CpNox2 is not essential for infection. Interestingly, Δcpnox2 mutants converted endophytic lifestyle to pathogenic lifestyle, and CpNox2 functioned in the infection process and moderates damage to the host 56 . In this study, the Nox1 of C. minitans is essential for mycoparasitism, conidiation and pigmentation, while deletion of Nox2 did not affect these bioprocesses. However, how Nox1 regulates these bioprocesses is necessary to be unraveled.
Rac1, a member of the Rho-family GTPases, widely exists in eukaryotes and in fungi as well. Sequence alignment of RAC1 homologs from fungi revealed significant conservation in amino acid composition and RAC1 from M. grisea could rescue the conidiation, parasitization and growth in ΔRac1 mutantof C. minitans (data not shown). Fungal Rac1 is crucial for the growth, virulence and development in many fungi, such as M. grisae, U. maydis, A. fumigatus, Claviceps purpurea and F. graminearum [57][58][59][60][61] . In M. grisea, MgRac1 is essential  for conidiogenesis, and contributes to the formation of appressorium and pathogenicity through activating its downstream factors: the PAK kinase Chm1 and NADPH oxidases 58 . In Candida albicans, Rac1 is an upstream regulatory factor of the MAP kinase Cek1 and Mkc1, but the control mechanism is still unclear 62 . In this study, the qRT-PCR assay demonstrated that the expression level of CmSlt2 was obviously decreased in ΔCmRac1. When CmSlt2 was over expressed in ΔCmRac1, the conidiation could be partially restored, further confirming that Rac1was an upstream regulatory factor of MAPK cascade signal pathway.
The fact that the Nox complex mutants and the MAPK cascade pathway mutants of fungi sharing highly similar phenotypes suggests that these two signal pathways are cross-linked. This study provides further evidence supporting this cross-link since CmNox1 could interact with CmSlt2, and CmRac1 could interact with CmNoxR. Lalucque et al. reported that PaNox1 acted on the PaASK1/PaMKK1/PaMpk1 MAPK module by promoting nuclear translocation of the PaMpk1 MAP kinase in P. anserina 63 . More recently, H 2 O 2 was found to induce the relocalization of a putative MAPK-activated protein kinase SrkA to nuclei and mitochondria under the presence of SakA in A. nidulans 53 . We further found that CmNox1 could regulate the nucleus location of a cell wall integrity-associated MAP kinase (CmSlt2). In ∆ CmNox1, CmSlt2 could not move into fungal nuclei, suggesting that Nox complex-derived ROS may function on the localization of CmSlt2. The Nox complex signal and MAPK cascade signal affect fungal gene expression globally to regulate complicated processes in physiology, pathogenicity and development. In S. macrospora, Nox1 affects the expression of genes involved in cytoskeleton remodeling, hyphal fusion, metabolism, and mitochondrial respiration 55 . In P. anserine, the expressions of 15% genes were modified in ΔPaMpk1, ΔPaMpk2 and ΔPaNox1, and about 1000 genes were regulated similarly in these three mutants 64 . We attempted to explain how the Nox complex signal and MAPK cascade signal regulate mycoparasitism by monitoring the expression levels of CmPks1, Cmg1 and CH1 in C. minitans. We found that expression of these genes was obviously enhanced when interacted with its host fungus S. sclerotiorum., while could not be induced in ΔCmNox1, ΔCmRac1 and ΔCmSlt2, indicating that these three genes were regulated by the Nox complex signal and MAPK cascade signal pathways, thus to affect parasitism. Mycoparasitism of C. minitans is a very complicated process. There are many other genes that are also involved in the mycoparasitism and remain to be investigated.
Briefly, we analyzed the function of Nox complex on mycoparasitism and conidiation of C. minitans, and we found that it is CmNox1, but not CmNox2, that is required for mycoparasitism and conidiation. We further found that CmNox1 interacts with MAP kinase CmSlt2 and effects the location of CmSlt2. Our finding suggests that Nox complex signal pathway and MAPK cascade signal pathway are cross-linked via CmNox1.

Methods
Strains and cultural conditions. The C. minitans wild-types train ZS-1 (CCAM 041057) was isolated from garden soil at Zhushan County, Hubei Province, P R China 65 . Strain Ep-1PNA367 was a virulent and virus-free strain of S. sclerotiorum, derived from a single ascospore of the hypovirulent strain Ep-1PN 66  Analysis of colony morphology, growth rate, conidiation and parasitic ability. Colony morphology was observed on PDA after incubating at 20 °C for 12 days. Observation of the pycnidial formation was performed by following the method described 14 . To characterize the biological properties of the mutants, growth rate, conidial production and parasitic ability were examined as described 13,14 . For all the transformants obtained, three individuals were examined (Table1).

ROS detection assay. Production of superoxide was evaluated with NBT using the method modified from
Chen et al. 58 . The wild-type ZS-1, mutants ΔCmNox1 and ΔCmNox2 were grown on PDA for 7 days. Mycelia were incubated in 0.05 M sodium phosphate buffer, pH 7.5, containing 0.05% (w/v) NBT (Sigma-Aldrich). After 1 h of incubation, the culture was fixed in ethanol to stop the reaction. The stained sample was examined with a compound microscope at × 400 magnification.
Isolation of CmNox1/CmNox2. A PCR primer pair 1F1/1R1 (Table S1) was designed based on the C. minitans genome database. CmNox1 was amplified from the ZS-1 genomic DNA by a 32-cycle PCR reaction (94 °C, 1 min; 58 °C, 1 min; 72 °C, 2 min), followed by a 10 min extension at 72 °C. The PCR product was cloned into the pMD18-T vector (TaKaRa) and confirmed by DNA sequencing. The cDNA of CmNox1 was isolated by RT-PCR from total RNA of C. minitans with the primer pair 1F1 and 1R1, followed by cloning into the pMD18-T vector and direct DNA sequencing. The same method was used to clone CmNox2 in C. minitans with the primer pair 2F1/2R1 (Table S1).
In order to construct the complementary vector of CmNox1, a 2.7 kb fragment containing the native promoter and ORF of CmNox1 was amplified by PCR with primer pair 19F1/19R1 (Table S1) and cloned into vector pneoP3300. The complementary mutant CmNox1-C was generated by introducing the vector into ΔCmNox1-6, followed by screening for neomycin resistance and RT-PCR confirmation.
cDNA of CmSlt2 was amplified by RT-PCR with primer pair 20F/20R (Table S1) and cloned into the HindIII/SmaI sites of pCIT vector, which contains the trpC promoter and terminator, and then the gene cassette was cloned into vector pneoP3300 to get the CmSlt2 over-expression vector pOVSlt2. The over-expression mutant OVS-Rac1-1 was generated by introducing pOVSlt2 into ΔCmRac1-79, followed by screening for neomycin resistance and qRT-PCR confirmation.
To tag CmSlt2 with the GFP, cDNA of CmSlt2 without stop codon was amplified by RT-PCR with primer pair 20F/20R (Table S1) and cloned into the HindIII/SmaI sites of pCIT vector. The GFP coding sequence was excised from plasmid pEGFP-1 with SmaI/BamHI, and the corresponding fragment was purified. Both the CmSlt2 and GFP coding sequences were cloned into vector pneoP3300 digested with XhoI to yield plasmid pSGFP1. Mutants were generated by introducing of pSGFP1 into ΔCmNox1-6 and the wide-type strain ZS-1, followed by screening for neomycin resistance. GFP signal was observed under a Nikon Eclipse 80i fluorescent microscope (Nikon, Japan).
Scientific RepoRts | 6:24325 | DOI: 10.1038/srep24325 DNA extraction and Southern blot analysis. The genomic DNA of the wild-type strain ZS-1 and generated mutants in this study was extracted according to the standard protocols 67 . Southern blot analysis was performed according to Gong et al. 17 . Genomic DNA (ZS-1 and CmNox1 mutants) aliquots of 15 μg were digested with HindIII, separated by electrophoresis on 0.8% agarose gel and transferred onto a Hybond N+ membrane (Amersham Pharmacia Biotech). Interior probe was amplified with the primer pair 30F and 30R (Fig. 2c, Table  S1), while the exterior probe was amplified with the primer pair 31F and 31R (Fig. 2c, Table S1). CmNox2 mutants were also confirmed by Southern blot, with the probe amplified using primer pair 32F and 32R.

Bimolecular fluorescence complementation experiments.
For bimolecular fluorescence complementation experiments (BiFC), CmNox1 and CmSlt2 were tagged with separated halves of YFP, based on Walter et al. 68 , with the following modifications. To fuse YFP-N to CmSlt2, CmSlt2 cDNA without stop codon was amplified with primer pair 7F and 7R and cloned into the SpeI/SmaI sites of pUC-SPYNE1. To fuse YFP-C to CmNox1, CmNox1 cDNA without stop codon was amplified with primer pair 8F and 8R and cloned into the SpeI/SmaI sites of pUC-SPYCE1. The two vectors were co-transformed into tobacoo (Nicotiana benthamiana). Leaf samples were collected 2 days after agro-infiltration. YFP Fluorescence signal was observed under a Nikon Eclipse 80i fluorescent microscope (Nikon, Japan).
In vivo Co-IP. Agrobacterium strain EHA105 carrying the TrpC-CmSlt2-GFP and TrpC-CmNox1-Flag expression vectors was coinfiltrated into C. minitans. The over-expression mutant was generated by screening for neomycin/hygromycin resistance. Mutant transformed with TrpC-CmSlt2-GFP and empty Vector-Flag was generated and used as a control. For in vivo Co-IP, 8-day-old mutant total protein was extracted in IP buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1% Triton, 1 mM EDTA, 1 mM MgCl 2 , and protease inhibitor cocktail). A 15 ul aliquot of anti-Flag agarose (Beyotime) was added to the samples, and the mixtures were incubated for 4 h at 4 °C with shaking. The immunoprecipitated proteins were washed three times with IP buffer and eluted with SDS sample buffer. Eluted sample were loaded on the protein gels for immunoblot analysis using anti-GFP antibody or anti-Flag antibody (Sigma).