Allium Sativum Leaf Agglutinin (ASAL) Endowed Enhanced Resistance Against Myzus Persicae under Both Constitutive and Phloem Specic Promoters

Globally, aphid, Myzus persicae is an economically signicant, polyphagous crop pest that feeds on more than 400 plant species and transmits more than 100 plant viruses. Aphid infestation is mostly managed by insecticides that cause heavy environmental contamination and insect resistance. Cloning of plant derived insecticidal genes to develop transgenic plants under suitable promoter is a promising technology. In the present study, ASAL (MN820725) was isolated from native garlic and cloned in plant transformation vector, pGA482 through Agrobacterium mediated tobacco transformation. PCR of genomic DNA of transgenic tobacco plants using gene specic primers conrmed the presence of asal gene of 546 bp. To detect the integration of gene Southern blot analysis was conducted that revealed stable integration of asal gene while, gene expression was analyzed through qRT-PCR that showed variable expression of asal gene in transgenic tobacco plants. Ecacy of asal gene was evaluated through aphid bioassay. Aphid bioassay revealed that transgenic tobacco lines LS-17, LS-20, LR-1, and LR-7 exhibited 100% aphid mortality and signicantly reduced the aphid population. These ndings suggested the potential of ASAL against aphids that can be further used against other notorious sap sucking pests.


Introduction
Insect pest control has become a challenge for sustainable agriculture. Physical and molecular defense of plants have evolved against biotic and abiotic stress. Meanwhile, insect pests have also adopted themselves against defense system of plants (Napoleao et al. 2018). Although morphological and chemical defense of plants alter the preference (host plant selection, feeding behavior) and performance plant-viruses as vector and secret honeydew that allow the growth of sooty molds on plant surface. Aphid feeding damages the strength of plants and stunt their growth. Growth of sooty mold covers the plant surface that retards the process of photosynthesis and distorts the plants ). In addition of short life cycle, female adults are capable to produce nymphs sexually, asexually or both (Dedryver et al. 2013). Aphid infestation triggers phosphorylation, calcium ux, and production of reactive oxygen species (ROS) that uctuate the production of phyto-hormone and interrupt the transcriptional regulation of plants (Liang et al. 2015;Shah et al. 2017).
Different control strategies based on physical barriers, pesticides, biotic agents and host-plant resistance have been used to combat sucking pests (Shukla et al. 2016). Transgenic technology is an economical and environment friendly approach to manipulate host plant resistance and incorporate insecticidal genes in plants (Arora and Sandhu 2017). Allium sativum leaf agglutinin (ASAL) is a mannose-binding lectin that has been reported in garlic (Allium sativum). It targets sucking insect pests and binds to their glycosylated receptors including alkaline phosphatase (ALP), aminopeptidase-N (APN), cadherin-like proteins, polycalins, sucrase, symbionin, crosses the hemolymph and deposits in the ovarioles and/or in fat bodies. These processes disrupt the membrane integrity, inhibit feed digestion and retard nutrient absorption that ultimately lead towards insect mortality (Upadhyay and Singh 2012). It has been reported that deposition of ASAL in ovarioles of aphid retards circulation of nutrients that could reduce aphid's fecundity up to 74%. Aphid fecundity is also reduced when ASAL binds with NADH quinone oxidoreductase (NQO), a key component of electron transport chain, insect defense response and gametogenesis (Roy et al. 2014). In present study, ASAL has been introduced in tobacco plants to confer resistance against aphids and other sucking insect pests. ASAL was isolated from garlic leaves and cloned in plant transformation vector, pGA482 for Agrobacterium mediated tobacco transformation. Expression of ASAL was achieved under 2X35S constitutive promoter and rolC phloem speci c promoter to speci cally target phloem feeders, aphids. Molecular characterization and aphid bioassay of transgenic tobacco plants revealed insecticidal effectivity of ASAL. These ndings could be useful to develop environment friendly sucking-pest resistant crop varieties.

Material And Methods
In-silico characterization of promoters and asal gene The cis-regulatory elements within the 2X35S constitutive promoter and rolC phloem speci c promoter were identi ed using PlantCARE software for comparative analysis (Lescot et al. 2002).
The nucleotide sequence of ASAL was translated through Swiss Bioinformatics Server (Artimo et al. 2012). Signal peptide and subcellular localization of protein was predicted by TargetP 1.1 Server (Emanuelsson et al. 2000). Molecular weight and isoelectric point of protein was computed using in-silico analysis (Gasteiger et al. 2005). Domain was predicted by online PROSITE scan tool (Sigrist et al. 2012 (Untergasser et al. 2012) to amplify full length asal gene from garlic. RT-PCR of 50 µl reaction mixture was conducted using proofreading DNA polymerase, pfu (2.5 U), and cDNA (1 µg) to amplify 546 bp full length asal gene using 10 µM of gene-speci c primer pair, ASAL-F3 and ASAL-R3 (Table 1). Reaction mixture was run in thermal cycler under following conditions; initial denaturation at 94° C for 5 min followed by 39 cycles of 94 °C for 1 min, 63°C for 1 min, 72°C for 1 min and nal extension of 72 °C for 5 min. Ampli ed gene was eluted using a Gel Extraction Kit (Cat No. K2100-12, Invitrogen) and cloned in PCR blunt cloning vector (Cat No. K2700-20, Invitrogen). Resulting clone was con rmed through restriction analysis and Sanger sequencing.
Full length asal gene was restricted from PCR blunt vector using HindIII and SmaI and cloned in pJIT163 having 2X35S promoter and CaMV terminator. Gene cassette, 35S-ASAL-CaMV was restricted from resulting plasmid pIT163-ASAL using SacI and EcoRV and cloned in plant transformation vector, pGA482. The resulting plasmid, pGA482-35S-ASAL was con rmed through restriction analysis.
2X35S promoter of pIJT163 was replaced with rolC promoter to clone full length asal gene under phloem speci c rolC promoter. Engineered gene cassette (rolC-ASAL-CaMV) was cloned in pGA482. The resulting plasmid, pGA482-rolC-ASAL was con rmed through restriction analysis.

Agrobacterium mediated tobacco transformation
Plasmids pGA482-35S-ASAL and pGA482-rolC-ASAL were separately, electroporated in Agrobacterium tumefaciens strain, LBA4404 and their cultures were developed. For stable transformation, leaf discs of Nicotiana tabacum (cv. Samsun) (Amaya 1997) were co-cultivated with engineered cultures of A. tumefaciens. and kept over the regeneration media containing MS salt and sucrose to develop transgenic tobacco plants..

Molecular analysis of tobacco plants
All putative transgenic tobacco plants were screened using gene speci c and construct speci c primer pairs (Table 1). CTAB (cetyl trimethylammonium bromide) method (Doyle and Doyle 1987) was used to isolate genomic DNA from leaves of tobacco plants expressing ASAL under 35S and rolC promoters, respectively. PCR reaction mixture of 25 µl was prepared using 100 ng of DNA, 12.5 µl of Dream Taq Green PCR Master Mix (2X) (Cat No. K1081, Thermo Scienti c), and 1 µl of each primer (10 µM). Gene speci c primers were used to con rm the presences of asal gene while construct speci c primers were used to detect the promoter-gene fragment (35S-ASAL or rolC-ASAL) in transgenic tobacco plants. PCR reaction mixture was run in thermal cycler at 94°C for 5 min followed by 35 cycles of 94°C for 1 min, 63°C for 1 min, 72°C for 1 min and nal extension of 72°C for 5 min. The PCR ampli ed products were checked on 1% agarose gel to screen transgenic tobacco plants.
Integration and copy number of transgene (asal) was detected in T 0 tobacco lines through Southern blotting. For this genomic DNA was extracted from the transgenic tobacco expressing ASAL under 35S and rolC promoters. DNA (100 µg) samples were restricted using EcoRI at 37°C overnight and then subjected to the electrophoresis using 0.8% agarose gel at 25 volts overnight. Fractionated DNA fragments were transferred to the positively charged nylon membrane. Nylon membrane was crosslinked with UV linker (0.240 J/cm 2 ) (Strata linker) and hybridized with DIG (digoxigenin) labelled probe. Nylon membrane was washed using Dig Wash and Blocking Buffer Set (Cat No.11585762001 Roche) and signals were developed using color substrate NBT/BCIP. qRT-PCR for gene expression analysis qRT-PCR was conducted for relative gene expression analysis of asal gene in T 0 transgenic tobacco lines.
RNA was isolated from the 3-4 leaf stage (45 days old) transgenic and non-transgenic tobacco plants using SV Total RNA Isolation System (Cat No. Z3101, Promega). Primers were designed using Primer3 program. Concentration of primers were optimized, and qPCR reaction mixture was prepared containing, cDNA (200 ng), Power SYBR Green PCR Master Mix (Thermo Scienti c), gene speci c primers (qASAL-F5, qASAL-R5) and 18S rRNA as internal control (Table 1). Three replicates/sample were loaded on 96 well plate to run in Quantstudio 6 Real-time PCR system (Thermo Fisher Scienti c) using following conditions;

Statistical analysis
Signi cant difference of relative gene expression was determined between transgenic and non-transgenic tobacco plants using ANOVA followed by LSD. Mann-Whitney U-tests was used to calculate signi cant difference of mortality and fecundity between aphid feeding on transgenic and non-transgenic tobacco.

Results
Cis-regulatory elements of promoters PlantCARE software identi ed several motifs in 35S and rolC promoter sequence. Phytohormone responsive elements like auxin, methyl jasmonic acid and abscisic acid responsive elements were identi ed in both 35S and rolC promoter. Light responsive elements were frequently found dispersed throughout the rolC promoter. The predicted regulatory elements of both promoters have been illustrated in Table 2.
In-silico analysis of asal gene Signal peptide of ASAL consist of 30 residues and belongs to the secretary protein with Reliability Class 2. The theoretical weight of ASAL was estimated at 19.25 kDa with an isoelectric point (pI) 9.05. PROSITE tool predicted bulb-type lectin domain in ASAL that spanned over 31-140 residues that is D-mannose speci c domain and contains disul de bond between 59 and 83 residues. ConSurf results estimated the evolutionary conservation of amino acid positions in ASAL protein molecule based on the phylogenetic relations between homologous sequences (Fig. 1A-B). The server results were predicted in the range of scale 1-9, where scale 1 is highly variable and scale 9 is highly conserved. Besides the conservation scale, the server also predicts whether the amino acid is functional or structural or exposed or buried (Fig.  1C). Phylogenetic analysis revealed the homology of ASAL protein among different species of Allium and other related families of plant species (Fig. 1D).

Aphid bioassays
Data of aphid bioassay revealed entomotoxic effect of ASAL expressed under 35S promoter or rolC promoter. Survival and fecundity of adult aphids reduced during feeding on transgenic tobacco. Tobacco lines expressing ASAL under 35S showed considerable level of resistance against aphids than nontransgenic tobacco (Fig. 7A-7C). Tobacco lines LS-17 and LS-20 showed 100% aphid mortality while 80-90% mortality was showed by LS-15, LS-18, LS-21 and LS-25 (Fig. 7D). Different transgenic lines showed lethal effects on aphids during different time interval. LS-18 showed aphid mortality after 48 hours while LS-21 showed aphid mortality after 72 hours. Expression of ASAL in transgenic tobacco under 35S promoter also reduced the population of aphids and retarded the aphid fecundity. Transgenic tobacco lines showed up to 63% reduction in aphid fecundity than non-transgenic tobacco plants (Fig. 7E).

Discussion
Aphids are deleterious crop pest that drain off phloem sap, transmit plant viruses and secret honeydew that allow the growth of molds (Eid et al. 2018). Expression of insecticidal genes in transgenic plants is an effective strategy to control sap sucking insect pests. Lectins have emerged as promising and ecofriendly insecticidal proteins for the development of insect resistance crop plants. Lectins can tolerate proteolytic effect of insect mid gut and confer entomotoxic effect against sap sucking insect pests, aphids (Caccia et al. 2012). In present study we have evaluated the e cacy of a mannose binding lectin, ASAL under 2X35S constitutive promoter and rolC phloem speci c promoter.
Choice of promoter is an effective criterion to achieve higher expression levels of a gene in transgenic plants (Hu et al. 2003). In the present study, we used 2X35 and rolC promoter to drive the expression of asal gene in transgenic tobacco plants. In silico study detected different cis-acting elements in both promotes. Presence of core transcriptional, metabolism, light responsive, temperature and phytohormones related cis-elements in 2X35S promoter showed its constitutive nature as studied earlier Allium sativum leaf agglutinin (ASAL) is a mannose-binding lectin having potent insecticidal activity against sucking complex as well as bollworms (Ghosh et al. 2016). Previous studies have reported 50% hydrophobic nature of signal peptide of ASAL that allows its synthesis on endoplasmic reticulum and follows the secretory pathway (Damme et al. 1998). Our results of ConSurf model (Fig. 1A-D) prediction also indicated 50% part of signal peptide is exposed (hydrophilic) while 50% part is embedded (hydrophobic) while subcellular localization of ASAL through TargetP 1.1 indicated its secretory nature. In-silico analysis of asal gene predicted N-terminal signal peptide and B-type lectin domain. The degree to which an amino acid position is evolutionarily conserved is strongly dependent on its structural and functional importance. Thus, conservation analysis of positions among members from the same family . Phylogenetic analysis revealed that ASAL and its orthologue are from important spice, ornamental and medicinal plants. So, it is inferred that ASAL lectin might have evolutionary importance for plant's own defense against insect-pests. Moreover, due to distribution of this ASAL and other lectin proteins in mostly edible and medicinally important crops may show its safe history. This indicates that ASAL and its homologs are used safely to develop transgenic plants. The phylogenetic analysis revealed that the translated asal is related to B-lectin family and present across different families in the plant kingdom and may have a widespread defensive role against pest-complex. A bulb lectin super-family (Amaryllidaceae, Orchidaceae and Aliaceae) contains a ~115 residue long domain. Each bulb-type lectin domain consists of three sequential β-sheet subdomains (I, II, III) that are inter-related by pseudo three-fold symmetry.
Tobacco lines expressing 35S-ASAL and rolC-ASAL were developed through Agrobacterium mediated transformation. ASAL expression levels in transgenic tobacco plants were considerably high as indicated by aphid bioassays. Tobacco lines, expressing ASAL under 35S promoter and rolC promoter showed considerable aphidicidal effects. It is reported that mannose binding lectin, Galanthus navlis agglutinin (GNA) showed equivalent insecticidal effects against sap sucking pests under both constitutive and phloem speci c promoter (Rao et al. 1998). Earlier ndings suggested that ASAL under 35S constitutive promoter and rolC phloem speci c promoter showed less or more equivalent rate of mortality and reduction in fecundity of sap sucking insect pests (Chakraborti et al. 2009). Our study also supported the similar results as we found that expression of ASAL in transgenic tobacco caused signi cant aphid mortality and reduced aphid fecundity during aphid bioassays. Maximum tobacco lines (LS-17, LS-20, LR-1and LR-7) showed up to 100% aphid mortality and reduced aphid fecundity up to 78%. In the present study, the expression of ASAL was achieved under 35S promoter and rolC promoter to evaluate the e ciency of both promoters. It was found that irrespective to the type of promoters the expression and e ciency of ASAL was equivalent effective against aphids. Previous studies reported that expression of transgene under constitutive promoter could be high and/or equivalent to phloem speci c promoter (Nakasu et al. 2014). It is also reported that mannose binding lectins expressing under both constitutive and phloem speci c promoter showed equivalent insecticidal activity against sap sucking pests (Saha et al. 2007). Therefore, we conclude that ASAL expressed under both 2X35S and rolC promoters could be used for transformation of other crops. Furthermore, ASAL could be used in combination with other insecticidal genes for durable and sustainable insect pest control.  Tables   Table 1 Primers used for cloning, PCR and qRT-PCR  Table 2 Cis-acting elements associated with 35S promoter and rolC promoter