Molecular and Biochemical Characterization of Cotton Epicuticular Wax in Defense against Cotton Leaf Curl Disease

1. Background
    Plant viruses are major hindrance in yield improvement and productivity of plant products. Viruses that belong to family Geminiviridae, are economically important and transmitted by the members of the phylum Arthropoda (1).
    Cotton plants are naturally affected by many stresses from which 75% are biotic (2). Among these pathogens,Cotton leaf curl virus (CLCuVand its cognate CLCuBuVand CLCuMB) is a common source of tension for cotton growersespecially in Pakistan. CLCuV genome consists of a single stranded DNA particle i.e. DNA-A along with each of its associated DNA satellites, called alphasatelliteand betasatellite (3).
    The first and foremost physical barrier in plant pathogen interaction is epicuticular wax (4). This layer not only hinders the bacteria and fungi, but also create a first line of defense against insects (5). For instance, in wax deficient pea mutants the aphid spendsmore time (6).  Wax can be defined as  a polyester matrix of hydroxyl- and hydroxyl epoxy fatty acids C16 and C18 long (cutin) embedded and overlaid with epicuticular wax.
    The Asiatic G. arboreum is resistant to CLCuV (7). Our hypothesis was to investigate that whether the wax plays a critical barrier in transmission of CLCuV by whitefly (Bemisiatabaci) in this plant. In 2009, a wax deficient mutant (GaWM3) of Asiatic G. arboreum with 50% less wax wasproduced (8).

2. Objectives
    The aims of the present study was (1) to quantify the cuticular waxes and determine the biochemical composition of wax mutant GaWM3in comparison with G. arboreum, G. hirsutum and G. harknessii, and (2) to determine the CLCuV titer and its correlation with quantity and composition of waxes through feeding of whiteflies on plants.
 
3. Materials and Methods

3.1. Plant Materials
    Seeds of G. hirsutum  less waxy and  susceptible to CLCuV, G. arboreum, “desicotton” resistant to CLCuV with more epicuticular wax, G. harknessii, more waxy like  and susceptible to CLCuV were planted along with wax deficient mutant of G. arboreum (GaWM3) in pots as well as in field.Upward or downward curling of the leaves, thickened veins and growth of plants was noted in inoculated and non-inoculated plants as indicated by Khan et al. (9).

3.2. CLCuV Titer Evaluation
    Viruliferous whiteflies (100) were incubated overplants. Field trials have been conducted under natural infection condition with uncharacterized CLCuV isolates. However, it was found that CLCuBV was more dominant in the field than CLCuMB Primers were designed for alphasatellite (FR873751.1), betasatellite (HF567946.1) and DNA-A (X98995.1). The primers and probe (5’Fam and 3’Tamra) were designed from coat protein of DNA-A, C1 region of beta-satelliteand Rep gene of alpha-satellite (Table 1) using “Genscript” website software (https://www.genscript.com/ssl-bin/app/primer). The experiment was performed in 3 replicates. The reaction mixture (25 mL) contained 150 mg of plant DNA, 2.5 mL 10× PCR buffer (Fermentas), 2.5 mL of 2 mM dNTPs, 1.5 mL of  MgCl2 (Fermentas) 1 mL of 10 pmol.mL-1 each forward and reverse primers (Table 1) and 0.5 mL of 5UTaqDNA-polymerase (Fermentas). The PCR was initiated at 95ºC for 5 min, followed by 35 cycles of 95ºC for 30 s, 59ºC for 30 s, and  72ºC for 30 sec with final extension at 72ºC for 10 min. The concentrations of the viral particles were calculated through Real Time-PCR using standard curve through known standards of DNA-A, alphasatellites and betasatellites.
 
3.3. Wax Quantification
    The isolation of plant epicuticular wax was performed according to “Decoction” method (10) and leaf surface area was calculated with Adobe Photoshop (11).  Thetotal isolated wax from each plant was converted into µg and divided by total leaf surface area (in cm2).

3.4. Determination of Biochemical Composition of Epicuticular Wax
    Gas chromatograph mass spectrometry:wax samples (1 mg) in 3 replicates were dissolved in hexane and passed through impregnated carbon filter to remove any impurities. Internal standard, tetracosane (10 mg.mL-1) was added to the testing samples prior to analysis. From the wax samples, 2 mL was taken and injected into the column at 50ºC and condition was held for 2 min. The samples were desorbed by increasing the temperature by 40°C/min to 200°C, 2 min at 200°C, 3°C/min to 310°C, and 30 min at 310°C. The Helium gas was used as the carrier and the gas flow was maintained at 2 mL.min-1. The quantitative composition of the mixtures was studied by capillary GC (Agilent; 30 m HP-1, 0.32-mm i.d. df = 1 mm) and flame ionization detection under the same GC conditions as above but Helium (carrier gas) inlet pressure was programmed for 50 kPa at injection, held for 5 min, raised with 3 kPa.min-1 to 150 kPa and held for 40 min at 150 kPa. Single compounds were quantified against the internal standard by manually integrating peak areas (12). Components were identified by the help of NIST library, 2005 (13).

3.5. Whitefly Feeding Assay
     Two week old seedling of plants (i.e. G. arboreum, GaWM3, G. hirsutumand G. harknessii) were placed into Hoagland’s solution (14) with 1% Nile Blue (Sigma Aldrich). The whiteflies (Bemisiatabaci) were incubated on plants for 3 days and observed under microscope (Zeiss, Imager A1) to observe the color of Nile Blue dye in their gut.

4. Results

4.1 Detection of CLCuV
    Symptoms: The plants were exposed to whiteflies in random in field trials and 100 whiteflies per plant were incubated in greenhouse tests. The symptoms of cotton leaf curl disease appeared on G. hirsutum, G. harknessii and GaWM3 but not on G. arboreum. The typical symptoms of upward or downward curling of the leaves and thick enation were appeared on G. hirsutum and GaWM3 (Figure 1). The CLCuV components (alphasatellite, betasatellite and DNA-A) were quantified by real time PCR. The mean numbers of molecules per microliter of alphasatellite in betweengreenhouse and field samples were 5.9×108, 4.8×107 and 4.6×107 for G. hirsutum, GaWM3 and G. harknessii, respectively.Whereas no alphasatellite was detected in G. arboreum (Figure 2A). Betasatellites were determined as 7.2×108, 3.6×107 and 3.8×107 molecules.mL-1 in G. hirsutum, GaWM3 and G. harknessii, respectively. Similarly, betasatellite was not detected  in G. arboreum (Figure 2B). The copy numbers of DNA-A in G. hirsutum, GaWM3 and G. harknessii were 8.7×108, 6.6×107 and 6.3×107 molecules.mL-1, respectively. Again, DNA-A was not detectedin G. arboreum (Figure 2C). In experimental plants, G. hirsutum: GaWM3: G. harknessii: G. arboreum, the ratio of a-satellitewas 270:24:23:0 for alphasatellite, for betasatellite was 360:18:19:0, andfor DNA-A was 290:22:21:0, respectively.

4.2. Epicuticular Wax per Unit Area
    Maximum wax per unit area was obtained from G. arboreum (183 mg/cm2) as compared to its mutant that had 95 mg/cm2. In contrast, G. hirsutum and G. harknessii had130 mg/cm2 and 146 mg/cm2, respectively.

4.3. Biochemical Composition of Epicuticular Wax
    Gas chromatograph mass spectrometry of plants (G. arboreum, GaWM3, G. hirsutum and G. harknessii (Figure 3A-D, respectively) was carried out to determine the biochemical composition of wax and their quantitative values. The chemical compounds were identified by comparing their retention time in the NIST mass spectra library, 2005 (13).
    The top 3 compounds that were dominant in G. arboreumare suspected to be (1) 25.6%heptadecanoicacid, 16-methyl-, methyl ester (2) 14.1% phenol, 2,5-bis [1,1- dimethyl] and (3) 10.12% 1,2-benzenedicarboxylic acid, diisooctyl ester. The dominant compounds in wax of GaWM3 were suspepcted to be (1) 18%1,2,- benenedicarboxylic acid, diisooctyl ester (2) 14% octadecane, 1-|2-(hexadecyloxy)ethoxy|- (3) 12%7,9-Di-tet-butyl-1-oxaspiro (4, 5) deca-6, 9-diene-2,8-dione and (4) 11% nonadecane having percentage. The three major compounds found in the wax of G. hirsutumwere (1) 25% 1,2-benzenedicarboxlic acid, diisooctylester (2) 21% nonadecaneand (3)14% phenol, 2,5-bis (1,1-dimethyletyhly)- with percentage of, .Lanceol, cis- and  caryophyllene were the two major wax compounds found in G. harknessii, having the percentage of 45% and 36%, respectively. Comparison of wax biochemical composition of experimental plants is shown in (Table 2).
    
4.4. Whiteflies Feeding Assay
    Collected whiteflies on G. arboreum, similar to the negative control did not show any gut coloring (Figure 4 A,B), while on the other 3 plants, gut color was observed (Figure 4 C-D).
 
5. Discussion
    Here, a cotton wax mutant (GaWM3) next to 3 other wild type cotton species were analyzed to establish the role of wax in resistance against insects. The plant having less wax is more susceptible to insects, G. arboreum wax deficient mutant (GaWM3) was found susceptible to CLCuV (Figure 2) as opposed to the wild type (7).
    The concentration of the isolated waxes were  183, 146, 130 and 95 mg/cm2 in G. arboreum, G. harknessii, G. hirsutum and GaWM3, respectively. The concentration of the wax was in accordance with the report of Bondada et al. (15) i.e. from 70 mg/cm2 to 154 mg/cm2 from normal condition to stress conditions in cotton.
    The results of virus symptoms appearance were in accordance with (16) and (17). The role of betasatellite is well-defined in suppressing the phyto-immune system that ultimately results in development of severe viral symptoms (18, 19).  Our data support this hypothesis that increase in quantity of betasatellite results in increase of symptoms and vice versa. The positive correlation was found in the severity of the symptoms and titer of betasatellite particles along with DNA-A (Figure 1).
     The ratio of different organic compounds varies in the epicuticular wax. Hydrocarbons, alcohols and acids were the major compounds found in the wax of red vine (Brunnichia ovata) and trumpet creeper plants (Campsis radicans) (20). In addition to these classes of compounds, esters, phenols and other aromatic compounds were also found in this study.Themost dominant compounds were esters in G. arboreum, GaWM3 and G. hirsutum (25.6%, 18% and 25%, respectively) and lanceol, cis (45%) was dominant in G. harknessii.
    The comparison of wax components of GaWM3 and G. arboreum clearly demonstrated that the following six organic compounds were only presentin G. arboreum: 3-trifluoroacetoxytetradecane, 2-piperidinone, n-[4-bromo-n-butyl], 4-heptafluorobutyroxypentadecane,silane, trichlorodocosyl-, 6-octadecenoic acid, methyl ester, and heptadecanoic acid, 16-methyl-, methyl ester,may create unique features in its wax and may be involved in its resistance against transmission of CLCuV (Table 2). The whitefly feeding assay also suggested that the quantity as well as quality of the wax has its role in feeding of whiteflies (Figure 4).

6. Conclusions
    The characterization of cotton epicuticular wax and its role in transmition of CLCuV by whiteflies to plants were demonstrated. It was found that 50% reduction in wax (in leaves of GaWM3) made it possible for the whiteflies to transmit the virus and to develop the relevant symptoms. It is concluded that wax act like barrier in hindering the CLCuV transmission in cotton. Moreover, quantities as well as chemical composition of wax had impacts on  feeding behavior in whiteflies and transmission of CLCuV.