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Journal of Horticultural Science, Studies on the Xingiang isolate of cauliflower mosaic virus. Acta Microbiologica Sinica, Plants also must rely on their own immune systems. If a plant can utilize its ability to interfere with gene expression then virions will stop being replicated inside of the host. The plant is able to recognize foreign mRNA and stop gene expression before the plant continues producing for the virus [16]. There is no cure for cauliflower mosaic virus.
Once a plant is infected, then it is too late. Prevention is the only way to prevent agricultural or gardening losses. Since cauliflower mosaic virus is usually spread by aphids, so any sort of pest control can be beneficial in reducing infection.
Netting or pest control products may keep virus carrying insects at bay. Since this virus can be transported through any opening in plants such as abrasions or cuts, disinfecting tools, equipment and anything that contacts plants will reduce infections.
It is best to get rid of infected plants immediately to reduce exposure to plants nearby. Also, get rid of seeds coming from infected plants because CaMV could be transmitted to plant offspring.
Keeping weeds away from the farm or garden can also eliminate virus harboring organisms. Finally, there are varieties of plants with resistance to caulimoviruses that are unable to get infected in the first place [9] [10].
Cauliflower mosaic virus has a very important function in biotechnology. It is because of its efficient promoter that is used to produce cloned genes, that it can be used to create transgenic plants that can take use of this efficiency. In order to do this, genes can only be inserted in the minor coding regions or regions not necessary for virus production which include open reading frame II and open reading frame VII. If done correctly, the production of progeny viruses will not be affected.
A use of this is to insert a dihydroxyl folatereductase gene so that the infected plant will be resistant to methotrexate which otherwise is very toxic to plants [15]. Producing transgenic plants is done in order to gain benefits such as higher yield, resistance, quality and efficiency which would be hugely impactful to farmers who have been plagued by mosaic viruses and had to throw away a large percentage of their crop [11].
There are some limitations to using cauliflower mosaic virus in the production of transgenic plants. These limitations include the small margin for error when inserting genes only in open reading frame II and open reading frame VII [11]. It is also feared that trangenetic plants could induce the evolution of new pararetroviruses similar to how bacteria evolve to be resistance to antibiotics [16].
Regulatory cis proteins are necessary in interacting which transcription factors to initiate gene expression. The 35S promoter in Cauliflower Mosaic Virus, is used in combination with other 35S subdomains in other plants in order to produce tissue specific gene expression. These other 35S subdomains are different in other plants which means the same combinations of 35S subdomains produce different genes depending on the host plant.
The resulting transgenic plant that hosts the Cauliflower Mosaic Virus 35S promoter is expected to differ developmentally in its tissues than the non transgenic plant. Different combinations of 35s subdomains will then be expected to change the development of other plant tissues. Having quick transgenic plant development and easily observed tissue changes, this process is able to be repeated very efficiently and provide more data involving the changes occurring from the CaMV 35s promoter [17].
Deletion analysis has been used in order to identify the specific functions of 35S promoter regions.
Some regions found in tobacco callus and leaf tissue such as the to region is heavily involved in the activation of transcription.
This exemplifies how different 35S regions play a role in gene expression which can be used to confer tissue specific gene expression in a multitude of plant species. For example, domain A was found to be involved in root or embryonic root development as well as the meristematic portion of the plant stem and domain B was found to be involved in the upper portions of the plant.
These domains are also found in some cases to only be active when they are working synergistically. In tobacco seeds, sub-domain B1 does not change gene expression and change tissue development in seedling growth. However, paired with domain A, differing tissue development and gene expression can be detected.
B2 sub-domain paired with the A domain does not affect seed and seedling development at all. Instead, this combination changes in leaf phloem, stem and mature root development.
In contrast with the effects of the CaMV promoter in tobacco, petunia plants express genes differently while still using the same 35S promoter. Similarly in tobacco, petunia also requires interaction of the A and B domains in order for many of the B sub-domains to activate gene expression. In contrast, petunia relies on interaction among cis elements unlike tobacco plants.
Moreover, two ethylene response mutants, etr and ein , exhibited decreased susceptibility to CaMV infection [ 36 , 37 ]. In this scenario, whether P6 can interfere with chromatin modification of minichromosome against host defenses through defense-related hormone pathways deserves further investigation. This study investigated how the multifunctional protein CaMV P6 interfered with the deposition of histone acetylation on viral minichromosome through dysfunction AtHD2C against host defenses.
Moreover, HD2C acted as a positive defense regulator during the CaMV infection by regulating the level of histone acetylation on the viral minichromosome. In addition, P6 overexpression lines were hypersensitive to the ABA and NaCl, which was similar to that of the hd2c mutants. Nicotiana benthamiana plants at the stage with leaves were used for bimolecular fluorescence complementation assay and co-immunoprecipitation assays.
The CaMV-infected leaf extract solution was then used to inoculate the 8-leaf stage Arabidopsis by rubbing the leaves with carborundum. The control group was mock-inoculated in a similar way using phosphate buffer with non-infected leaf extract to create similar mechanical damage.
BiFC experiments were carried out as described previously [ 40 ]. Agrobacterium cells were resuspended to OD 0. The Agrobacterium cell solution was injected into the lower epidermis of N. After 2 days of cultivation, the lower epidermis of N. For protein purification, cells were centrifuged and resuspended using lysis buffer 20 mM Tris-HCl pH 7. The pull-down assay was performed as previously described [ 41 ]. Then the GST beads were added into the mixture and incubated for an additional 2 h.
After incubation, beads were washed six times, and the pulled-down proteins were detected by western blotting. Tumefaciens strain, and then Agrobacterium cells were injected into tobacco leaves. After 2 days of incubation, protein extracts were obtained by lysing the tobacco leaves using co-immunoprecipitation buffer 50 mM Tris-HCl, pH 7.
GTX antibodies. Quantification of the band intensities on the western blot was performed using the Image J software. After 3-day stratification, the seeds were germinated in a long-day condition growth chamber.
Germination rates were measured after 2 days, with a criterion that radicle protrudes from episperm. To measure the leaf survival rate of seedlings under high salinity, we transferred 5-day-old seedlings to the medium containing mM NaCl. After 5 days of treatment, the leaf survival rate of seedlings was measured. The gene-specific primer sequences are listed in Supplemental Table S1. The ChIP assay was performed as described previously with slight modification [ 42 ].
All samples were powdered in liquid nitrogen and incubated in M1 buffer 10 mM phosphate buffer pH 7. After incubation, the mixture was filtrated using a four-layer Miracloth and centrifuged. The pellet was suspended, centrifuged, and washed three times using M2 buffer 10 mM phosphate buffer pH 7. ACTIN7 was used for normalization.
The resulting transformants grew on a selective medium with 5 mM 3-Amino-1, 2, 4-triazole indicated that HD2C interacted with P6 Figure 1 a. We also conducted in vitro pull-down analysis. To further prove the interaction in vivo, we carried out co-immunoprecipitation Co-IP and bimolecular fluorescence complementation BiFC assays. Benthamiana Figure 1 c. P6 interacts with HD2C in vivo and in vitro. The YFP fluorescence signal was observed on confocal microscopy.
It is reported that a histone H4 deacetylase, OsHDT70, could regulate the level of histone H4 acetylation on the promoter region of defense-related genes to negatively regulate innate immunity in rice, providing insight into the roles of histone deacetylase in biotic stress responses [ 24 ]. We carried out a viral inoculation experiment using the wild-type Col-0 as a control and recorded symptom development.
As shown in Figure 2 a, although the symptoms and severity of the infected Col-0 and hd2c-1 plants were similar, both exhibited chlorosis, vein-clearing and leaf mosaics, half of the hd2c-1 mutant plants showed disease symptoms at Symptomatic plants were measured and shown as percentages at different days after infection.
In order to propagate in the host, plant DNA viruses like geminiviruses and CaMV use host histone to pack their DNA genome into a chromatin-like structure called minichromosome [ 32 , 43 ].
Furthermore, it has been proven that both chromatin-activation modification and chromatin-repressive modification are present in geminivirus minichromosome [ 44 ]. On account of the genome of CaMV, which can be packed by host histone into minichromosome after infection, we tested whether AtHD2C could influence the viral gene expression on minichromosome through histone deacetylation.
We conducted a chromatin immunoprecipitation and qPCR to test the level of histone acetylation on the CaMV minichromosome. As shown in Figure 3 a,b, the depositions of histone acetylation on 35S and 19S promoter regions of viral minichromosome were significantly increased in hd2c mutants than the wild-type, suggesting that AtHD2C may have a role to inhibit the viral gene expression on minichromosome through histone deacetylation. AtHD2C inhibits the viral gene expression on minichromosomes through histone deacetylation.
The experiment was repeated three times with similar results. Our results have shown that the levels of H3K9K14ac and H3K4me 3 are both globally increased in P6 overexpression lines compared with the wild-type Figure 4 b.
Similar results were also observed in CaMV infected plants compared with control Figure 4 c. With the lastingness of infection, the amount of viral minichromosome increased, and the levels of H3K9K14ac and H3K4me 3 increased gradually Figure 4 c.
P6 dysfunctions HD2C. The loading control immunoblots of Actin were used for normalization. The loading control immunoblots were shown as the levels of Actin. To confirm our hypothesis and study the effect of biotic stress caused by the CaMV virus on the abiotic stress, we investigated the seed germination rate of P6 overexpression lines under the ABA and NaCl stress. Overexpressing P6 showed similar phenotypes to hd2c-3 , with lower germination rates than the wild-type under the ABA and NaCl stress Figure 5 a,b.
We also investigated the leaf survival rates of P6 overexpression lines under high salinity stress. Leaves left green were measured as the percentage of leaf surviving.
The leaf survival rates of P6 overexpression lines were lower than the wild-type, similar to hd2c-3 Figure 5 c,d. Five-day-old seedlings were transferred to the medium containing mM NaCl. Luo et al. We further detected the expression of these ABA-regulated genes in CaMV infected plants compared with non-infected plants.
Total RNA was extracted from leaves with systemic symptoms. P: the promoter, E: the first exon. AtHD2B and OsHDT, members of HD2-type HDAC, have been reported to control the expression of biotic stress response genes by directly binding to them and modulating histone acetylation levels, playing vital roles in the host defense against bacteria and fungi pathogens [ 23 , 24 ].
Recently, it was reported that a list of histone acetyltransferases HAT showed elevated expression when the inoculation with RNA virus occur in Triticum aestivum [ 45 ]. In this work, we found that histone deacetylase HD2C functions as a positive regulator in defense response to CaMV infection, and its regulation of histone deacetylation on DNA-viral minichromosome was interfered by interaction with CaMV P6 protein.
The interaction between viral protein and HDACs had been previously demonstrated. This result gave insight into a possibility that P6 causes HD2C to dysfunction, leading us to speculate that the overexpression of P6 might have a similar phenotype with hd2c mutants. To verify our conjecture, we first detected the global level of H3K9K14ac and H3K4me 3 in P6 overexpression lines and found that the level of these modifications increased Figure 4 b.
Consistent with the hypothesis, seed germination rates and the leaf survival rates of the P6 overexpression lines were lower than the wild-type when treated with ABA and NaCl, similar to hd2c mutant Figure 5. It is, therefore, possible that the dysfunction of HD2C caused by P6 may also influence the function of HDA6, leading to similar phenotypes between overexpression lines and hda6 mutant. For example, both P6 overexpression lines and hda6 mutant exhibited late flowering [ 48 , 49 ].
Pathogen-induced histone modification is a hotspot of research. Histone acetylation and deacetylation were involved in host defense responses to virus pathogens [ 28 , 29 , 30 , 45 , 50 ].
HD2C is a well-studied and characterized HDAC, whose function is established not only in plant development but in plant abiotic and biotic stress responses [ 14 ].
However, the involvement of HD2C in biotic stress responses was limited to bacterial and fungal pathogens, with unknown functions in antiviral response and the underlying mechanisms. Our results have shown that hd2c-1 was hypersensitive to CaMV infection, and the amount of viral DNA and the expression of the viral transcript were higher in hd2c mutants than the wild-type, indicating a positive regulatory role of HD2C in antiviral response Figure 2.
As a deacetylase, HD2C repressed gene expression by removing the acetyl group from histone. Thus, the underlying mechanism that HD2C positively resisting against CaMV is possible to repress viral gene expression through histone deacetylation on viral minichromosome. Our ChIP results indicated that the levels of histone deacetylation on 35S and 19S promoter regions of viral minichromosome were significantly increased in hd2c mutants than in the wild-type Figure 3 b.
It is increasingly recognized that viral infections can facilitate host adaptation to abiotic stress, raising a novel idea of using viral infection as a probe to uncover the unknown molecular mechanisms of the abiotic stress response. An example was that virus infection by a list of RNA viruses significantly enhanced plant tolerance to drought and freezing stress, with a probable mechanism that the level of osmoprotectant and antioxidant were increased in infected plants [ 51 ].
Further research revealed that the drought tolerance induced by Cucumber mosaic virus CMV , an RNA virus, was caused by the 2b viral protein and the 2b-transgenic plants exhibited hypersensitivity to ABA-mediated inhibition of germination [ 52 ].
Another report demonstrated that plants subjected to more viral virulence showed higher tolerance to drought than plants subjected to lesser viral virulence [ 53 ].
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