Transient expression of SbDhr2 and MeHNL in Gossypium hirsutum for herbivore deterrence assay with Spodoptera litura

Spodoptera litura (Lepidoptera: Noctuidae), commonly known as tobacco cutworm or cotton leafworm, is a polyphagous pest which causes considerable damage to cotton (Gossypium hirsutum) and other crops. Herbivore-induced defence response is activated in plants against chewing pests, in which plant secondary metabolites play an important role. Dhurrinase2 (SbDhr2), a cyanogenic β-glucosidase from Sorghum bicolor, is the key enzyme responsible for the hydrolysis of dhurrin (cyanogenic β-glucosidic substrate) to p-hydroxymandelonitrile. Hydroxynitrile lyase (MeHNL) from Mannihot esculanta catalyses the dissociation of cyanohydrins to hydrogen cyanide and corresponding carbonyl compound, both enzymes play a pivotal role in plant defence mechanism. SbDhr2 and MeHNL genes were expressed individually and co-expressed transiently in cotton leaves. We examined the feeding response of S. litura to leaves in the choice assay. The S. litura population used in this study showed better feeding deterrence to leaves co-expressing both genes compared with the expression of an individual gene. Our results suggest that co-expression of SbDhr2 and MeHNL genes in cotton leaves demonstrate feeding deterrence to S. litura. Engineering cyanogenic pathway in aerial parts of cotton would be an additional defence strategy against generalist pests and can be enhanced against specialist pests.


Background
Spodoptera litura (Lepidoptera: Noctuidae) commonly known as tobacco cutworm or cotton (Cheng et al. 2017) leafworm is a polyphagous pest, causes considerable damage to cotton (Gossypium hirsutum) and various other crops (Xue et al. 2010;Bragard et al. 2019). Failure to control S. litura and its resistance to various insecticides, lead to humongous economical loss (Ahmad and Gull 2017;Fand et al. 2015). Plant secondary metabolites, play a direct role in plant defence response (War et al. 2012;Schaller 2008) and in the adaptation of plants to abiotic/biotic stresses (Akula and Ravishankar 2011;Bartwal et al. 2013;Gleadow et al. 1998;Rosenthal and Berenbaum 1992). Engineering plant metabolic pathways would be a feasible alternative defence strategy against generalist insect pest. In two-component defence system, β-glucosidases and cyanogenic glucosides are separated by different subcellular compartments (Saunders and Conn 1978;Thayer and Conn 1981;Kesselmeier and Urban 1983;Poulton and Li 1994). In plant physiology, βglucosidases play diverse roles (Morant et al. 2008), and more than 2 500 species of plants contain Cyanogenic glucoside (Panter 2018). Metabolic engineering of the whole cyanogenic pathway in different plants has been reported for insect herbivory deterrence (Franks et al. 2006;Blomstedt et al. 2016;Tattersall et al. 2001;Bak et al. 2000).
Transient gene expression in cotton using virus-induced gene silencing (VIGS) vector (Becker 2013) (TRV: Tobacco Rattle Virus) has already been performed (Li et al. 2018;Gao et al. 2011;Pang et al. 2013) for functional genomic studies. Expressing cyanogenic pathway enzymes in upland cotton can help to develop insect-pest resistant cotton varieties.

Plant materials
Seeds of cotton (G. hirsutum) were sown in pots containing peat moss and kept at 23°C, 200 μmol . m -2. S − 1 light, 65% relative humidity with 16 h/8 h day-night photoperiod in a growth room. After the emergence of four to five true leaves, cotyledons were used for infiltration.
MeHNL gene digested with EcoRI and SacI was ligated in TRV2 to get the second recombinant plasmid pTH2 (Fig. 1b). Verification of clones was done by restriction enzyme digestion.
Then infiltrations were performed in 20 plants for each suspension, at the abaxial side of cotyledons with a needleless syringe (Gao et al. 2011; Senthil-Kumar and Mysore 2014) (Fig. 2).
Gene detection in non-infilterated leaves using PCR DNA was isolated from non-infiltrated leaves by the cetyl trimethyl ammonium bromide (CTAB) method (Healey et al. 2014). Then PCR analysis was performed using primers listed in Table 2 for SbDhr2 and MeHNL genes. PCR positive leaf samples were further analysed for protein expression.

Western blotting for confirmation of gene expression
Total protein was extracted from sorghum, cassava, control plants and PCR positive non-infiltrated cotton leaves (Fig. 3). For Western blotting (Trans Blot Turbo transfer system) 40 μg of total protein was transferred on Amersham Hybond-P 0.45 PVDF blotting membrane, as per manufacturer's instructions. Amersham Hybond-P 0.45 PVDF, a 0.45 μm pore size polyvinylidene difluoride (PVDF) hydrophobic membrane, is used with standard colorimetric and chemiluminescent detection methods for proteins. Blots were probed with primary polyclonal antibodies raised in rabbit for SbDhr2 and MeHNL proteins, detected with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG antibody, and chemiluminescence was performed using Pierce™ ECL Western blotting substrate as per manufacturer's instruction. Membranes were exposed to Xray film, then developed and fixed.

S. litura herbivory deterrence assay
Three settling preference choice tests (Krothapalli et al. 2013) were conducted in petri-plates containing a control leaf and a leaf transiently singly expressing and coexpressing SbDhr2 and MeHNL genes on wet germination paper (Table 3). Plates were kept at room temperature at a relative humidity of 68% and 16 h/8 h day-night photoperiod. Five of the third instar larvae of S. litura were released in the centre of each plate after being starved for 3~4 h, and the settling preference of insects was measured after every 24 h period till the 8 th day. Each choice assay was replicated four times. The number of insects on each leaf was used to measure the settling preference and t-test was performed with GraphPad prism-8 for insect count. Mean weight of the five larvae before feeding and post feeding for 2 days (48 h) and on the 8th day was recorded in all three set of tests.

Cyanogenic capacity (HCNc) in infiltrated leaves
Amount of hydrogen cyanide released per unit time is measured as Cyanogenic Capacity (HCNc) (Hay-Roe et al. 2011). In this experiment, we were interested in temporal comparisons of HCNc in three different

Validation of plasmid for transient expression
After verification of plasmids, pTH2 and pTD2 by restriction enzyme digestion, a 790 base pairs (bp) MeHNL gene fragment was released from pTH2 vector. A 2.3 kilobase (kb) SbDhr2 gene along with its PEPC promoter gene was released as a restriction digestion product from linearized pTD2 vector (Fig. 4). Colony PCR screening of A. tumefaciens (LBA4404) using gene specific primers confirmed the presence of pTH2 and pTD2 vectors (Fig. 5). Colony PCR gave amplification product of 770 bp for MeHNL gene (Fig. 5a) and a 2.3 kb amplicon of SbDhr2 gene along with its promoter (Fig. 5b).

Screening of genes and expression in non-infiltrated cotton leaves
PCR analysis confirmed the presence of MeHNL (Fig. 6a) and SbDhr2 genes (Fig. 6b). Fifteen leaf samples were screened for presence of MeHNL and SbDhr2 genes, of which 13 samples were PCR positive for each gene. PCR results of negative control leaves confirmed the absence of either gene. Western blot analysis of total protein from five randomly selected PCR positive leaves confirmed the presence of MeHNL (29.3 kDa) (Fig. 7a) and SbDhr2 (62 kDa) (Fig. 7b) proteins bands. Un-infiltrated and empty vector infiltrated leaves of cotton served as negative control.

Insect herbivory measurements
A two-tailed t-test (GraphPad prism-8) for the insect number on a leaf after 48 h indicated that a greater number of pests preferred settling on control leaf (Fig. 8a, c, e) as per observation. On the 8th day, settling preferences were recorded with no significant difference (P > 0.05) in plants singly expressing MeHNL and SbDhr2 genes with respect to the control leaves (Fig. 8b, d). Significant difference (P < 0.05) (Fig. 8f) was recorded in settling preference on the 8th day, where MeHNL and SbDhr2 genes were co-expressed compared with the control leaf. Settling preference results substantiate visual observation of damage caused by pests on control and independently expressed MeHNL and SbDhr2 proteins in leaves and was nearly homogeneous on the 8th day ( Fig. 9a~b) with no significant difference, whereas leaf samples co-expressing both SbDhr2 and MeHNL proteins demonstrated a better feeding deterrence till the 8th day (Fig. 9c~d) as observed. The damage caused in co-expressed infiltrated leaf is far less compared with control and independently expressing proteins. Mean weight of five S. litura larvae before feeding was 0.020 mg, and the mean weight gained by larvae post feeding on all three sets of tests was recorded after 2 days (48 h)  and on the 8th day (Table 4). There was no significant difference observed in weight gained by larvae (Fig. 10).

Cyanogenic capacity (HCNc) in leaves
Cyanide released from transiently expressed positive leaves was measured per unit time (Ballhorn et al. 2010; Alonso-Amelot and Oliveros-Bastidas 2005) to determine cyanogenic capacity (HCNc). No remarkable difference in MeHNL infiltrated or SbDhr2 infiltrated leaves was observed, whereas light colour change was observed in leaves co-expressing both enzymes after 9~10 h (Table 5).

Sources of HCN in cotton plants and its detoxification pathways
Cyanogenic glycoside (CNglcs), also known as specialized secondary metabolites, is derived from amino acids, Ltryosine, L-valine, L-leucine, L-isolucine, L-phenylalanine with oximes and cyanohydrins as important intermediates.   Tiny amount of hydrogen cyanide is produced by all plants as a product or a co-product of a biosynthesis pathway. There are four reactions/ metabolic pathways that would liberate hydrogen cyanide in cotton on the basis of enzyme predictions (http://ptools.cottongen. org), i.e., ethylene biosynthesis I (plants) pathway (Xu and Zhang 2015), linustatin bioactivation (Schmidt et al. 2018;Jørgensen et al. 2005), neolinustatin bioactivation (Forslund et al. 2004;Lai et al. 2015) and vicianin bioactivation pathway (Mizutani et al. 2007).
HCN in plants is detoxified by two pathways. In the first pathway, HCN is converted to 3-cyano-L-alanine (Machingura et al. 2016), and is further metabolized to Lasparagine and L-aspartate (Asparagine pathway); in the second pathway, thiosulfate sulfurtransferase (rhodanese) (Nakajima 2015;Steiner et al. 2018) catalyses the conversion of thiosulfate and cyanide to thiocyanate and sulfite.

Heterologous expression of SbDhr2 and MeHNL in aerial parts of cotton
We have successfully demonstrated that transient coexpression of SbDhr2 and MeHNL could help to deter S. litura from feeding on cotton leaves. Higher expression of SbDhr2 compared with MeHNL gene was observed in Western blotting, which can be attributed to the choice of promoters, the use of PEPC (Matsuoka et al. 1994) and 2X CaMV 35S (Samac et al. 2004;Christensen et al. 1992;Weeks et al. 1993). According to hydrogen cyanide release detection by using Fiegl-Anger test paper, no colour change was observed in control and leaf tissue independently infiltrated with pTD2 or pTH2, whereas light colour change was observed after more than 9 h in leaf tissue samples co-infiltrated with pTD2 & pTH2 construct.

Lessons learned from transient expression
These findings along with the previous work (Pant et al. 2016) indicate that α-hydroxynitrile glucoside exists in G. hirsutum. There are possibilities that cyanide detoxification route/pathway (Gleadow and Moller 2014;Machingura et al. 2016;Ting and Zschoche 1970;Zagrobelny et al. 2004;Miller and Conn 1980;Sun et al. 2018;Nielsen et al. 2016;Pičmanová et al. 2015) is more active in cotton.

Conclusion
This study was conducted to investigate whether transient expression of cyanogenic pathway enzymes in aerial parts of cotton protects plants against herbivory by S. litura. The results presented here clearly support the finding that transient co-expression of cyanoamino acid metabolism pathway enzymes can deter S. litura from feeding on cotton leaves. It has also demonstrated that strong green tissuespecific promoter of enzyme/transgene expression is a prerequisite for enhancing HCNp in cotton. These findings extrapolate novel opportunities for metabolic engineering of cyanogenesis in G. hirsutum, for which detailed knowledge of metabolic cross-talk, cyanogenic glucoside synthesis, transport, regulation and degradation is a prerequisite. Engineering cyanogenesis in cotton can be envisioned as an additional pest control strategy.