Construction of an Infectious DNA Clone of Grapevine Geminivirus A Isolate GN and Its Biological Activity in Plants Analyzed Using an Efficient and Simple Inoculation Method

The pathogenicity of grapevine geminivirus A (GGVA), a recently identified DNA virus, to grapevine plants remains largely unclear. Here, we report a new GGVA isolate (named GGVAQN) obtained from grapevine ‘Queen Nina’ plants with severe disease symptoms. The infectious clone of GGVAQN (pXT-GGVAQN) was constructed to investigate its pathogenicity. Nicotiana benthamiana plants inoculated with GGVAQN by agroinfiltration displayed upward leaf curling and chlorotic mottling symptoms. A simple, quick, and efficient method for delivering DNA clones of GGVAQN into grapevine plants was developed, by which Agrobacterium tumefaciens cells carrying pXT-GGVAQN were introduced into the roots of in vitro-grown ‘Red Globe’ grape plantlets with a syringe. By this method, all ‘Red Globe’ grape plants were systemically infected with GGVAQN, and the plants exhibited chlorotic mottling symptoms on their upper leaves and downward curling, interveinal yellowing, and leaf-margin necrosis symptoms on their lower leaves. Our results provide insights into the pathogenicity of GGVA and a simple and efficient inoculation method to deliver infectious viral clones to woody perennial plants.


Introduction
Viral diseases in viticulture lead to serious reductions in yield quantity and quality.With the development of high-throughput sequencing technology, new viruses infecting grapevines have been identified.To date, more than 80 different viruses from 17 families and 34 genera have been identified in grapevines [1,2].Grapevine geminivirus A (GGVA) was first reported in 2017 using high-throughput sequencing [3].Since its initial characterization, GGVA has also been reported in China [4], South Korea [5], New Zealand [6], and India [7].GGVA is a monoparticle, single-stranded circular DNA virus belonging to the genus Maldovirus in the family Geminiviridae.The complete genome of GGVA ranges from 2903 to 2907 nucleotides (nt) in length [8], including two open reading frames (ORFs) on the viral sense strand encoding V1 (coat protein) and V2 (putative movement protein), and four ORFs on the complementary strand encoding C1 (replication-associated protein), C2 (transcriptional activator protein), C3 (replication enhancer), and C4 (host-activated protein) [3].
Plants 2024, 13, 1601 2 of 10 Grapevine and other woody perennials are recalcitrant to mechanical inoculation.The main approaches for delivering infectious clones into grapevine plants include vacuumassisted agroinfiltration [2,9,16] and agro-drenching [10,15,18].However, these methods are complex and time-consuming, especially for the latter method, which requires more than 20 days to establish a viral infection.In addition, the two methods result in relatively low survival and infection rates for the treated plants [2].Therefore, the development of an efficient and simple experimental system to launch grapevine infections is needed.
In the present study, we constructed an infectious DNA clone of GGVA isolate QN (pXT-GGVA QN ), which was isolated from a grapevine 'Queen Nina' plant with severe disease symptoms, including malformation, upward curling of the leaf margins, vein clearing, and reduced leaf size.The biological activity of an infectious clone of GGVA QN was analyzed in N. benthamiana and grapevine plants.In addition, we provide a simple, time-saving and highly efficient inoculation method to deliver an infectious clone of a virus into woody perennial plants, including grapevine.

Construction of an Infectious Clone of GGVA Isolate QN
The recombinant plasmid pCE3-GGVA QN , containing the full genome of GGVA QN , was used as a template.The full genome of GGVA QN (2905 nt) was 100% identical to the isolate we obtained from the grapevine 'Marselan' (GGVA ML , GenBank accession no.PP397181) (Supplementary Figure S1a), and shared 96.72% to 99.70% sequence identity with previous reported isolates.A phylogenetic tree was constructed based on the whole genome sequences of GGVA isolates from GenBank using the neighbor-joining method.We found that GGVA QN is very close to the isolate reported by Sun et al. [8] (Supplementary Figure S1b).
To construct an infectious clone of GGVA QN , two fragments covering 1.2× viral genome sequence amplified from pCE3-GGVA QN and the linearized vector pXT1 (JN029690) [19] were assembled into circular plasmids (Figure 1), which were then transformed into competent Escherichia coli DH5α cells.Positive clones were identified by polymerase chain reaction (PCR), enzyme digestion, and sequencing.The recombinant plasmid (pXT-GGVA QN ) was transformed into Agrobacterium tumefaciens GV3101.
result in relatively low survival and infection rates for the treated plants the development of an efficient and simple experimental system to laun infections is needed.
In the present study, we constructed an infectious DNA clone of GGV (pXT-GGVA QN ), which was isolated from a grapevine 'Queen Nina' plan disease symptoms, including malformation, upward curling of the leaf clearing, and reduced leaf size.The biological activity of an infectious clon was analyzed in N. benthamiana and grapevine plants.In addition, we pro time-saving and highly efficient inoculation method to deliver an infectio virus into woody perennial plants, including grapevine.

Construction of an Infectious Clone of GGVA Isolate QN
The recombinant plasmid pCE3-GGVA QN , containing the full genom was used as a template.The full genome of GGVA QN (2905 nt) was 100% id isolate we obtained from the grapevine 'Marselan' (GGVA ML , GenBank PP397181) (Supplementary Figure S1a), and shared 96.72% to 99.70% sequ with previous reported isolates.A phylogenetic tree was constructed based genome sequences of GGVA isolates from GenBank using the neighbor-jo We found that GGVA QN is very close to the isolate reported by S (Supplementary Figure S1b).
To construct an infectious clone of GGVA QN , two fragments cover genome sequence amplified from pCE3-GGVA QN and the linearized (JN029690) [19] were assembled into circular plasmids (Figure 1), whi transformed into competent Escherichia coli DH5α cells.Positive clones were polymerase chain reaction (PCR), enzyme digestion, and sequencing.The plasmid (pXT-GGVA QN ) was transformed into Agrobacterium tumefaciens GV  Diagram of the strategy used to build the infectious clone of grapevine geminivirus A isolate QN (pXT-GGVA QN ).Fragment 1, fragment 2, and the linearized pXT1 are marked in blue, yellow, and purple, respectively.The 5 ′ terminus of fragment 1 and the 3 ′ terminus of fragment 2 separately contain the T-DNA right border (RB) and left border (LB), marked in pink and green, respectively.MCS, multiple cloning site region; oriV, origin of vegetative replication; KanR, kanamycin resistance gene; trfA, trans-acting replication protein that binds to and activates oriV; term: NOS terminator; HDV-RZ, hepatitis delta virus ribozyme; 2 × 35S, two copies of the 35S promoter region.
2.2.Infectivity of GGVA QN in N. benthamiana Plants Six-week-old N. benthamiana plants were agroinfiltrated with the infectious clone of pXT-GGVA QN , and agroinfiltration of the empty vector pXT1 served as a control.At 7 days postinfiltration (dpi), the pXT-GGVA QN -infiltrated leaves exhibited upward curling of the leaf edge, and symptoms of upward leaf curling together with chlorotic spots were observed on the upper systemically infected leaves at 21 dpi, whereas the control plants were asymptomatic (Figure 2a).All 37 N. benthamiana plants (10-13 plants, three repeats) were systemically infected with pXT-GGVA QN , as evidenced by PCR and Western blot assays showing the presence of GGVA QN in the first systemically infected and upper new developing leaves of all GGVA QN -inoculated plants at 3 and 6 dpi, respectively (Figure 2b).These results indicated that pXT-GGVA QN can successfully infect N. benthamiana plants and elicit upward leaf curling and chlorotic spot symptoms.
Plants 2024, 13, 1601 3 of 10 yellow, and purple, respectively.The 5′ terminus of fragment 1 and the 3′ terminus of fragment 2 separately contain the T-DNA right border (RB) and left border (LB), marked in pink and green, respectively.MCS, multiple cloning site region; oriV, origin of vegetative replication; KanR, kanamycin resistance gene; trfA, trans-acting replication protein that binds to and activates oriV; term: NOS terminator; HDV-RZ, hepatitis delta virus ribozyme; 2 × 35S, two copies of the 35S promoter region.

Infectivity of GGVA QN in N. benthamiana Plants
Six-week-old N. benthamiana plants were agroinfiltrated with the infectious clone of pXT-GGVA QN , and agroinfiltration of the empty vector pXT1 served as a control.At 7 days postinfiltration (dpi), the pXT-GGVA QN -infiltrated leaves exhibited upward curling of the leaf edge, and symptoms of upward leaf curling together with chlorotic spots were observed on the upper systemically infected leaves at 21 dpi, whereas the control plants were asymptomatic (Figure 2a).All 37 N. benthamiana plants (10-13 plants, three repeats) were systemically infected with pXT-GGVA QN , as evidenced by PCR and Western blot assays showing the presence of GGVA QN in the first systemically infected and upper new developing leaves of all GGVA QN -inoculated plants at 3 and 6 dpi, respectively (Figure 2b).These results indicated that pXT-GGVA QN can successfully infect N. benthamiana plants and elicit upward leaf curling and chlorotic spot symptoms.
We then used a very simple and efficient method to deliver pXT-GGVA QN to grapevine plants.The roots of each in vitro-grown grapevine plantlet were directly inoculated with 0.5 mL of Agrobacterium tumefaciens cells carrying pXT-GGVA QN by
We then used a very simple and efficient method to deliver pXT-GGVA QN to grapevine plants.The roots of each in vitro-grown grapevine plantlet were directly inoculated with 0.5 mL of Agrobacterium tumefaciens cells carrying pXT-GGVA QN by syringe and then transplanted into a pot (Figure 3a).The empty vector pXT1 served as a control.A total of 18 plants were inoculated with GGVA QN in two repeat experiments (9 plants each), and 15 plants survived after transplanting.As shown in Figures 3b and 4a, about 2 months later, 'Red Globe' plants inoculated with GGVA QN showed chlorotic mottling on the upper leaves; symptoms of downward curling and necrosis of the leaf margins, and interveinal yellowing were also observed on the lower leaves.Five months later, the color of the interveinal tissues of the lower leaves of a few plants changed from yellow to red.All of these symptoms were absent in the corresponding control 'Red Globe' plants.All 15 surviving plants were systemically infected with GGVA QN , as confirmed by the presence of GGVA QN in the upper leaves detected by PCR and Western blot assays 1 month after inoculation (Figure 4b).These results demonstrated that 'Red Globe' plants systemically infected with GGVA QN display symptoms including chlorotic mottling, downward curling and necrosis of the leaf margins, and interveinal yellowing.Our results also indicated that the simple inoculation method developed in this study is efficient for launching the infection of grapevine.
Plants 2024, 13, 1601 4 of 10 syringe and then transplanted into a pot (Figure 3a).The empty vector pXT1 served as a control.A total of 18 plants were inoculated with GGVA QN in two repeat experiments (9 plants each), and 15 plants survived after transplanting.As shown in Figures 3b and 4a, about 2 months later, 'Red Globe' plants inoculated with GGVA QN showed chlorotic mottling on the upper leaves; symptoms of downward curling and necrosis of the leaf margins, and interveinal yellowing were also observed on the lower leaves.Five months later, the color of the interveinal tissues of the lower leaves of a few plants changed from yellow to red.All of these symptoms were absent in the corresponding control 'Red Globe' plants.All 15 surviving plants were systemically infected with GGVA QN , as confirmed by the presence of GGVA QN in the upper leaves detected by PCR and Western blot assays 1 month after inoculation (Figure 4b).These results demonstrated that 'Red Globe' plants systemically infected with GGVA QN display symptoms including chlorotic mottling, downward curling and necrosis of the leaf margins, and interveinal yellowing.Our results also indicated that the simple inoculation method developed in this study is efficient for launching the infection of grapevine.

Discussion
GGVA was found in samples from both grapevine 'Nagano purple' plants with leaf chlorotic ringspot symptoms and asymptomatic 'Black Beet' plants [3].Fan et al. [4] reported that most grapevine samples that were positive for GGVA were asymptomatic, except for a few samples with foliar ringspot symptoms.Similarly, Jo et al. [5] reported the detection of GGVA in all 16 individual grapevine plants representing 12 different cultivars, but only the cultivar Shine Muscat displayed obvious symptoms (leaf malformation, vein clearing, and yellowing).Infectious clones of three GGVA isolates-GGVA-17YM1 [8] and GGVA-76 and GGVA-93 [16]-all induced upward leaf curling and stunting symptoms in N. benthamiana plants, but grapevine (cultivars Colombard, Salt Creek, Cabernet Sauvignon, and Vaccarèse) plants infected with GGVA-76 or GGVA-93 did not display any obvious symptoms [16].Therefore, our knowledge of the pathogenicity of GGVA to grapevine plants remains largely unclear.
In this study, we constructed an infectious clone containing the full genome of GGVA QN to investigate its pathogenicity.N. benthamiana plants that were systemically infected with GGVA QN showed symptoms of upward leaf curling and chlorotic mottling (Figure 2a), which were somewhat different from the upward leaf curling and stunting symptoms observed by Sun et al. [8] and Kuo et al. [16].GGVA QN shares 99.52%, 97.32%, and 98.51% sequence identity with GGVA-17YM1, GGVA-76, and GGVA-93, respectively.The inconsistency may not be due to the sequence differences between GGVA QN and the

Discussion
GGVA was found in samples from both grapevine 'Nagano purple' plants with leaf chlorotic ringspot symptoms and asymptomatic 'Black Beet' plants [3].Fan et al. [4] reported that most grapevine samples that were positive for GGVA were asymptomatic, except for a few samples with foliar ringspot symptoms.Similarly, Jo et al. [5] reported the detection of GGVA in all 16 individual grapevine plants representing 12 different cultivars, but only the cultivar Shine Muscat displayed obvious symptoms (leaf malformation, vein clearing, and yellowing).Infectious clones of three GGVA isolates-GGVA-17YM1 [8] and GGVA-76 and GGVA-93 [16]-all induced upward leaf curling and stunting symptoms in N. benthamiana plants, but grapevine (cultivars Colombard, Salt Creek, Cabernet Sauvignon, and Vaccarèse) plants infected with GGVA-76 or GGVA-93 did not display any obvious symptoms [16].Therefore, our knowledge of the pathogenicity of GGVA to grapevine plants remains largely unclear.
In this study, we constructed an infectious clone containing the full genome of GGVA QN to investigate its pathogenicity.N. benthamiana plants that were systemically infected with GGVA QN showed symptoms of upward leaf curling and chlorotic mottling (Figure 2a), which were somewhat different from the upward leaf curling and stunting symptoms observed by Sun et al. [8] and Kuo et al. [16].GGVA QN shares 99.52%, 97.32%, and 98.51% sequence identity with GGVA-17YM1, GGVA-76, and GGVA-93, respectively.The inconsistency may not be due to the sequence differences between GGVA QN and the three GGVA isolates; rather, other factors, such as the different ages of the N. benthamiana plants used for inoculation and fertilization, may be responsible.
The pathogenicity of GGVA QN on grapevine was also investigated using in vitrogrown 'Red Globe' rooted plantlets.Upon GGVA QN infection, all 'Red Globe' plants exhibited symptoms including chlorotic mottling, downward curling, necrosis of the leaf margins, and interveinal yellowing 2 months after inoculation and transplanting (Figures 3b and 4a).The in vitro-grown 'Red Globe'-rooted plantlets used for inoculation assays were negative for GGVA, GLRaV-4 and its strains GLRaV-5, GLRaV-6, GLRaV-9, GLRaV-De, and GLRaV-Pr, and another 17 viruses reported in China.Therefore, the symptoms of 'Red Globe' plants were closely associated with GGVA QN infection.Our results differ from those reported by Kuo et al. [16], where neither GGVA-76 nor GGVA-93 induced obvious symptoms on grapevines 'Colombard', 'Salt Creek', 'Cabernet Sauvignon' and 'Vaccarèse' plants, although the virological condition of these plants was unclear.Lovato et al. [12] reported that grapevine plants infiltrated with the infectious clone of grapevine Algerian latent virus exhibited different symptoms in different cultivars.Therefore, the inconsistency may be due to the different grapevine cultivars used in the two studies.Future research investigating the pathogenicity of GGVA QN in different grapevine cultivars is warranted.
One important factor that hinders the research on viruses of woody perennial plants, including grapevine, is the lack of a reliable experimental system for delivering infectious clones.Two main approaches, vacuum-agroinfiltration [2,9,16] and agro-drenching [10,15,18], are currently used to deliver infectious clones into grapevine plants.However, they are complex and time-consuming, and the achieved rate of infection is relatively low.In this study, we directly introduced A. tumefaciens cells carrying the infectious clone pXT-GGVA QN into roots of grapevine plantlets by syringe followed by transplanting the plantlets into pots, and all surviving plants (15 out of 18) were systemically infected with GGVA QN .Therefore, we provide a very simple, quick, and efficient method for delivering infectious clones into grapevine plants with no vacuuming operation, taking only a few minutes to complete the infiltration, and resulting in a 100% infection rate.To our knowledge, the experimental protocol reported herein is the most effective system for achieving grapevine infection with infectious clones of a virus, potentially facilitating grapevine virology research.
In conclusion, an infectious clone of GGVA QN was constructed, and its pathogenicity in N. benthamiana and grapevine plants was investigated.The results presented here provide important insights into GGVA pathogenesis as well as a very simple and highly effective method for launching grapevine infection with the infectious clone of a virus.
In vitro-grown rooted grapevine plantlets were grown on woody plant medium supplemented with 30 mg mL −1 sucrose and 1 mg mL −1 indole-3-butyric acid.N. benthamiana plants were grown in individual pots containing commercial soil, and 6-week-old plants were used.All plant materials were grown at 26-31 • C with a 16-h light regime.

Construction of Infectious Clone of GGVA
The full genome of the GGVA QN isolate was PCR amplified from a sample collected from a grapevine 'Queen Nina' plant in Guangxi Province in Southwest China using a TOPO-Blunt Cloning Kit (Vazyme, Nanjing, China) with the primer pair GGVA-1678-F/GGVA-1678-R previously described [8].The PCR products were cloned into the pCE3 Blunt Vector provided by the kit, and the recombinant plasmid pCE3-GGVA QN was used as the template to construct the infectious clone of GGVA.
Construction of the infectious clone of GGVA QN was performed essentially as described previously [16], with modifications.A fragment (the linearized pXT1 without the sequence of the 35S promoter, multiple cloning site region, ribozyme, and NOS terminator) (Figure 1) was amplified using the binary vector pXT1 as template with primer pair F27/R27.Two fragments, each covering 0.6× viral genome sequence, were amplified from pCE3-GGVA QN using primer pairs F25/R25 and F26/R26, with F25 and R26 containing a 30-bp sequence homologous to the T-DNA right border (RB) and left border (LB) on pXT1, respectively.The three fragments were assembled into circular plasmids using NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs, Ipswich, MA, USA).Positive clones were identified by Hind III digestion (resulting in a 7197-bp fragment) and PCR with the primer pairs F25/R25 and F26/R26 for amplifying fragment 1 (1886 bp) and fragment 2 (1934 bp), respectively.Three positive clones were then sequenced to verify the correctness of the GGVA genome sequence.The recombinant plasmid pXT-GGVA QN was delivered into competent A. tumefaciens strain GV3101 (Weidi Biotechnology, Shanghai, China) using the freeze-thawing method according to the manufacturer's instructions.

Inoculation
A. tumefaciens GV3101 carrying the infectious clone pXT-GGVA QN was cultured to an optical density of 1.0 at 600 nm.Agrobacterium cells were harvested by centrifugation and resuspended in the infiltration buffer (10 mM MES/NaOH pH 5.8, 10 mM MgCl 2 , 150 µM acetosyringone).Agroinfiltration of N. benthamiana plants was performed essentially as described previously [20].
For inoculation of in vitro-grown grapevine plantlets, rooted 'Red Globe' plantlets were removed from the culture jar and washed to remove the agar adhering to their base.Plantlets with pruned roots and leaves were placed on moist filter paper.A 0.5-mL aliquot of A. tumefaciens GV3101 cells carrying pXT-GGVA QN (for each grapevine plantlet) was introduced into the main root bark with a syringe, and the plantlet was transplanted into a pot (10 cm diameter) containing commercial soil (Figure 3a).These pots were placed in plates filled with a shallow layer of water.About 3 days after transplanting, new buds were observed, indicating that they had survived.We used 8-13 plants, or plantlets, for each treatment in each experiment.
All inoculated plants were fertilized once a week with water-soluble fertilizer (Peters Professional).

RT-PCR and PCR
The virological condition of the in vitro-grown grapevine plantlets was detected by RT-PCR and PCR, and the presence of GGVA in inoculated plants was assessed by PCR.

Figure 1 .
Figure 1.Diagram of the strategy used to build the infectious clone of grapevine isolate QN (pXT-GGVA QN ).Fragment 1, fragment 2, and the linearized pXT1 are m

Figure 1 .
Figure 1.Diagram of the strategy used to build the infectious clone of grapevine geminivirus A isolate QN (pXT-GGVA QN ).Fragment 1, fragment 2, and the linearized pXT1 are marked in blue,

Figure 2 .
Figure 2. Systemic infection of GGVA QN in Nicotiana benthamiana plants.(a) Symptoms induced by GGVA QN .An arrow in the left panel indicates upward curling of the leaf edge.(b) PCR (left and middle panels) and Western blot (right panel) assays confirm the presence of GGVA QN in N. benthamiana plants.Lanes 1-7: samples collected 1 to 7 days postinfiltration (dpi), respectively.Upper newly developing leaves collected 6 dpi were used for detection of GGVA V1 protein by Western blotting with anti (α)-V1 antiserum.P and N: positive and negative controls with pXT-GGVA QN and distillation-distillation H2O (ddH2O) instead of DNA, respectively.CBB, Coomassie brilliant blue staining confirmed equal loading.

Figure 2 .
Figure 2. Systemic infection of GGVA QN in Nicotiana benthamiana plants.(a) Symptoms induced by GGVA QN .An arrow in the left panel indicates upward curling of the leaf edge.(b) PCR (left and middle panels) and Western blot (right panel) assays confirm the presence of GGVA QN in N. benthamiana plants.Lanes 1-7: samples collected 1 to 7 days postinfiltration (dpi), respectively.Upper newly developing leaves collected 6 dpi were used for detection of GGVA V1 protein by Western blotting with anti (α)-V1 antiserum.P and N: positive and negative controls with pXT-GGVA QN and distillation-distillation H 2 O (ddH 2 O) instead of DNA, respectively.CBB, Coomassie brilliant blue staining confirmed equal loading.

Figure 3 .
Figure 3.A simple, quick, and efficient method for delivering the infectious clone pXT-GGVA QN into grapevine plants.(a) Diagram of the procedure for agroinfiltration.Arrow indicates the sprouting buds after inoculation and transplanting.(b) Symptoms induced by GGVA QN in grapevine 'Red Globe' plants 4 months after inoculation and transplanting.

Figure 3 .
Figure 3.A simple, quick, and efficient method for delivering the infectious clone pXT-GGVA QN into grapevine plants.(a) Diagram of the procedure for agroinfiltration.Arrow indicates the sprouting buds after inoculation and transplanting.(b) Symptoms induced by GGVA QN in grapevine 'Red Globe' plants 4 months after inoculation and transplanting.

Figure 4 .
Figure 4. Systemic infection of GGVA QN in grapevine 'Red Globe' plants.(a) Symptoms in upper (left panel) and lower (middle and right panels) leaves of grapevine plants inoculated with pXT-GGVA QN .Symptoms were recorded 2 (left and middle panels) and 5 (right panel) months after inoculation, respectively.(b) Systemic infection by GGVA QN in grapevine 'Red Globe' plants was confirmed by PCR (left panel) and Western blot (right panel) assays.Samples were collected from three pXT-GGVA QN -inoculated and control plants 1 month after inoculation and transplanting, respectively.P and N: positive and negative controls with pXT-GGVA QN and ddH2O instead of DNA, respectively.GGVA accumulation was detected with α-V1 antiserum.CBB, Coomassie brilliant blue staining confirmed equal loading.

Figure 4 .
Figure 4. Systemic infection of GGVA QN in grapevine 'Red Globe' plants.(a) Symptoms in upper (left panel) and lower (middle and right panels) leaves of grapevine plants inoculated with pXT-GGVA QN .Symptoms were recorded 2 (left and middle panels) and 5 (right panel) months after inoculation, respectively.(b) Systemic infection by GGVA QN in grapevine 'Red Globe' plants was confirmed by PCR (left panel) and Western blot (right panel) assays.Samples were collected from three pXT-GGVA QN -inoculated and control plants 1 month after inoculation and transplanting, respectively.P and N: positive and negative controls with pXT-GGVA QN and ddH 2 O instead of DNA, respectively.GGVA accumulation was detected with α-V1 antiserum.CBB, Coomassie brilliant blue staining confirmed equal loading.