Analysis of the Protein Profile of Cassava Plantlets (Manihot esculenta Crantz.) Resistance to Fusarium Wilt Disease

Applied Biology Study Program, Faculty of Mathematics and Natural of Sciences, University of Lampung, Bandar Lampung, Lampung, Indonesia 35145 Biology Masters Study Program, Faculty of Mathematics and Natural of Sciences, University of Lampung, Bandar Lampung, Lampung, Indonesia35145 Chemistry Masters Study Program, Faculty of Mathematics and Natural of Sciences, University of Lampung, Bandar Lampung, Lampung, Indonesia 35145


Introduction
Cassava (Manihot esculenta Crantz.), is the third most important crop in the world and a source of food and tree income throughout the tropics. Cassava cultivation can provide sustenance to more than 500 million farmers (Eleazu et al., 2014;Amponsah et al., 2014). Cassava is an important food commodity in Indonesia and in the future this commodity will have a more strategic role in the lives of the people and the country's economy. Based on the area of harvest of food commodities, cassava ranks third after rice and corn, which are the three main sources of carbohydrates in the community (Fauzi et al., 2015). According to FAOSTAT (2019), Indonesia is the 6 th cassava producing country in the world after Nigeria, Congo, Thailand, Ghana and Brazil with a production of 14,586,693 tonnes. The center of cassava land in Indonesia is controlled by Lampung Province with production reaching 7,387,084 tonnes. This situation makes Lampung a third supplier of national cassava production from the national production of 21,801,415 tonnes (Badan Pusat Statistik, 2015).
One alternative way of controlling disease that is safe for the environment, among others, is using resistant varieties. The development of cassava varieties that are resistant to Fusarium oxysporum can be carried out by in vitro selection methods, namely culturing explants in the form of tissues or organs on medium containing selective concentrations of fusaric acid (Nurcahyani, 2017;Nurcahyani et al., 2016a;2016b;2019a;2019b;2020).
Fusaric acid is a metabolite produced by several species of fungi from the genus Fusarium. This acid can be toxic (concentrations more than 10 5 M) thus inhibiting growth and culture regeneration, but at non-toxic concentrations (below 10 6 M) it actually helps to induce phytoalexin synthesis, a plant response forms to inhibit pathogenic activity (Bouizgarne et al., 2006).
The use of fusaric acid as a selective agent in in vitro selection can produce mutant cells or tissues that are insensitive to fusaric acid, so that after being regenerated into plants, they can produce strains resistant to pathogenic infections. The identification of mutants or variants that are insensitive to fusaric acid by in vitro selection has been carried out, among others, on the Sphatoglottis plicata (Nurcahyani et al., 2016a;2016b), vanilla (Nurcahyani et al., 2012;Nurcahyani, 2017), Cassava (Nurcahyani et al., 2019a;2019b), Phalaenopsis amabilis (L.) Bl. (Nurcahyani et al., 2020).
Research on Induced Resistance cassava with fusaric acid has been conducted before and found indications of tolerant fusaric acid concentrations for the selection of resistant plantlets in vitro. Inoculation of Fusarium oxysporum (Fo) fungal isolates on resistant cassava plantlets was carried out in vitro, followed by DNA pattern analysis compared to controls. The output of this study, in the form of a cassava mutant with a new (specific) DNA band measuring 550 bp (OPA_1) and 300 bp (OPA_10), is predicted to be a candidate RAPD marker for cassava resistance to Fo. Based on the results of these studies, further study needs to be done to ascertain whether the new DNA strand is really a peroxidase protein that causes the cassava mutant to be resistant to Fo, namely by protein profile analysis. The special characteristics of cassava plantlet and related to resistance to fusarium wilt can be examined molecularly, that is, through the analysis of protein profiles using the SDS-PAGE method. Comparison of protein bands formed by electrophoretic separation can be carried out to identify the gene product produced during Manihot esculenta plantlets selected by fusaric acid.
Based on the above background, more in-depth research is needed on the Induced Resistance of cassava plantlet resistant to Fusarium wilt disease, so that ultimately the long-term goal of this research will be achieved, namely the acquisition of cassava mutant varieties that are resistant to Fusarium oxysporum.

Planting and Selection of Cassava Plants (Manihot esculenta Crantz.)
Planting plants in Murashige and Skoog (MS) medium in culture bottles and added with Fusaric Acid (FA) with concentrations of 0 ppm (control), 60, 80, 100 and 120 ppm. Each concentration was carried out 5 times and each replication consisted of 2 cassava plants in each culture bottle. Plant selection was carried out for 30 days. At the end of the fourth week he was assessed to determine the concentration of fusaric acid yielding LC50 for in vitro cassava crop selection.

Cassava Plantlets Protein Extraction
Protein extraction was carried out by counting 1 g of plantlet leaves with each added 300 μL of Phosphate Buffer Saline (PBS) (8.55 g/L NaCl, 1.33 g/L Na2HPO4.2H2O, 0.34 g/L NaH2PO4.H2O. L) with a pH of 7 as extraction buffer and added with a protease inhibitor, then crushed using a mortar and pestle until homogeneous. The crushed sample was centrifuged at 13,000 rpm for 2 sec. Supernatant containing crude protein was taken and stored at -20C (Maniatis et al., 1982).

Measurement of Protein Concentration
After the crude protein is obtained, measurement of the protein concentration in each sample is carried out. Protein concentration was determined using the Bio-rad method (Bio-rad Assay). Determination of protein concentration was carried out by taking 2 µL of protein samples using a micropipette plus 200 µL of Bio-rad dye and 798 µL of distilled water, then mixed by resuspension, then read by a spectrophotometer (Beckman, DU-65) at wavelength (OD 595 nm). The protein concentration is known through the equation of the standard BSA protein standard curve function (Maniatis et al., 1982).

Determination of the Molecular Weight of a Protein
Determination of the Molecular Weight (MW) of protein was carried out using the SDS-PAGE method according to Maniatis et al. (1982). Electrophoresis at a voltage of 100 volts is carried out for 1.5 to 2.5 h. Protein staining was carried out in a 0.10% solution of Coomasie Brilliant Blue, shaken with a shaker overnight. After staining, destaining is carried out to remove excess color by immersing the gel in a destaining solution (50 mL distilled water, 40 mL methanol, 10 mL glacial acetic acid) until the gel becomes clear with separate bands from each other. The gel was then stored in 10% glacial acetic acid and then dried with a plate kit. The protein bands formed in the gel after electrophoresis were determined by their molecular weight (kD). The molecular weight of the sample protein at each migration distance is obtained by extrapolating every distance of the desired sample protein band at the 2 migration distance of the protein marker bands that flank the sample protein band in question, so that a log of molecular weight is obtained, then the molecular weight of the protein band in question can be known.
To detect the presence of new (specific) proteins, it was done by comparing the protein profiles of Cassava leaflets that were not affected by fusaric acid with cassava plantlets induced by fusaric acid.

Results and Discussion
In this study, using cassava plantlet (Manihot esculenta Crantz.) In vitro and treated with various levels of fusaric acid. The fusaric acid used in this study contained five different concentration levels, namely 0 ppm (control), 60, 80, 100 and 120 ppm. The results of the selection of cassava plantlets that have been induced using fusaric acid with various different concentration levels are presented in Fig. 1.
Based on Fig. 1, visually, the plantlets in each treatment varied, especially seen from the poor morphological characters of the plantlets, namely small size, shoots and roots that were slightly formed and some were not formed at all. The visual appearance of the plantlets which were originally green turned pale green and brown (browning) after being treated with fusaric acid In browning-resistant planlets it occurs only at the base of the planlet in contact with the medium and does not extend to the ends. Plantlets that do not withstand browning quickly expand to all parts of the plantlet.  Kuźniak (2001) stated that fusaric acid can affect the elimination of O2through quinone reduction which induces brown color and is thought to be associated with phenol transformation. The results of the phenol oxidation reaction are brown, so that the plantlets that have undergone phenol oxidation appear brown. If this oxidation reaction takes place continuously, the brown color will spread and diffuse into the medium and affect the growth of other plantlets that are cultured with the browning plantlets.
The specific character of Cassava plantlets and related to resistance to Fusarium oxysporum can be examined molecularly, namely through protein profile analysis using the SDS-PAGE method.
In principle, genes (DNA fragments) are transcribed into mRNA in the cell nucleus. Furthermore, the triplet base codon in mRNA is translated by the ribosome into amino acids. From this set of amino acids, a specific protein is formed. If there is a change in base and or structure in DNA/RNA, it is called a mutation which can be in the form of addition, deletion and substitution. As a result of this change in base, the expressed protein would certainly be different from those that did not undergo mutation. This concept will be discussed in Cassava's research with fusaric acid stress for resistance to Fusarium oxysporum. Plants treated with Fusaric acid will activate genes, including peroxidase, glucanase and chitinase genes (Saravanan et al., 2004).
The protein profile was obtained after the crude extract protein (concentration about 10 μg) was running by electrophoresis in 1D vertical gel (SDS-PAGE) for 2 h, with a voltage of 90 volts. The protein banding pattern formed in the mutant candidates turned out to be different bands or bands compared to the control. This occurred in all samples both at fusaric acid treatment concentrations of 60, 80, 100 and 120 ppm (Fig. 2).
Based on Fig. 2, it was found that the expression of a new protein band with a molecular weight of about 98 kD and a missing protein band with a molecular weight of 65 kD was found in the cassava Manihot esculenta Crantz. plantlet treated with 120 ppm fusaric acid stress. From the protein profile analysis, it indicated that M. esculenta plantlets treated with 120 ppm fusaric acid gave a different band than the control and fusaric acid treatment with a concentration of 60, 80 and 100 ppm.
This shows that fusaric acid triggers the expression of peroxidase genes in the Cassava plantlets, so that their activity is higher and the protein bands are more clearly stained. Based on this, it is suspected that there has been a mutation in the promoter so that the band with molecular weight approximately 98 kD can be indicated as a marker for Fusarium oxysporum resistant cassava plantlets. According to Gunanti et al. (2010), the protein band thickness of the SDS-PAGE results illustrates the high and low concentration of a protein contained in the test sample. The induced resistance of cassava plantlets with fusaric acid treatment, one of the possibilities was due to the activation of the peroxidase gene which encodes the peroxidase enzyme and plays an important role in resistance to Fusarium oxysporum.
Research conducted by Ye and Ng (2002) on French beans has successfully isolated protein peroxidase and produced bands with molecular weight approximately 37 kD. In sorghum plants infected with Fusarium moniliforme, induction of resistance protein with molecular weight around 18 and 30 kD and predicted as protein peroxidase (Kumari et al., 2006). A protein with molecular weight around 18.9 kD has also been found by Ye and Ng (2009), which is an antifungal induction protein from Japanese takana seeds (Brassica juncea var. integrifolia). Meanwhile, Ye et al. (2011), found an antifungal protein with molecular weight around 30 kD in red cabbage (Brassica oleracea). Nurcahyani et al. (2016a), in their research on vanilla (Vanilla planifolia) which was induced by fusaric acid and infected with Fusarium oxysporum, causing resistance induction with a molecular weight of about 18 kD and predicted as protein peroxidase.
Genes need the right time and conditions to be expressed in the cycle of growth and development of plants. Entering a new stage of development, plants need the expression of several genes to produce proteins that play a role in every metabolic reaction in cells. In this case, it may be endogenously, the gene encoding the peroxidase enzyme is present in plantlets, but is only expressed when the condition is stressful, in this case fusaric acid. The expression of this peroxidase enzyme appears as a mechanism of resistance to fusaric acid stress and is also a resistance to Fusarium (Bouizgarne et al., 2006).

Conclusion
The character of cassava (Manihot esculenta Crantz.) plantlets which are resistant to Fusarium oxysporum can be proven molecularly, namely through protein profile analysis. The protein band (molecular weight approximately 98 kD) on SDS-PAGE 1D indicated the resistance to M. esculenta plantlets against Fusarium wilt disease. The protein with a molecular weight of about 98 kD is predicted to be a protein peroxidase, which plays a role in resistance to F. oxysporum. The perspective to ensure that the protein bands with molecular weight of about 98 kD is protein peroxidase, it is necessary to isolate the protein, analyze its amino acid base by aligning it with the protein peroxidase sequence of other species, analysis of transient gene expression and/or Western blot analysis, this will be done at the next research stage.