A total of thirteen 5′- (CsVMV1-CsVMV13) and three 3′- (CsVMVR1-CsVMVR3) end deletion fragments were PCR amplified (using primers listed in Supplementary Table S1) adding EcoRI and HindIII sites at the 5′- and 3′- ends, respectively. The PCR amplified products were subsequently cloned in the EcoRI and HindIII sites of pUC119 sequencing vector and subsequently sequenced and analyzed to check their integrity. The fragments were finally sub-cloned into protoplast and plant expression vectors pUCPMAGUS and pKYLX71GUS (Schardl et al., 1987; Dey and Maiti, 1999), respectively.

For the generation of chimeric promoter construct MUASCsV8CP, the UAS of MMV (MUAS; 335 bp, -297 to -38) and the proximal domain of CsVMV8-Flt (CsV8CP; 381 bp, -215 to +166) promoter were PCR amplified using appropriate primers (Supplementary Table S1) containing overhangs of EcoRI and HincII at the 5′- end and SmaI and HindIII at the 3′- end. The PCR amplified fragments were cloned into the corresponding sites of pUC119 following a previously published protocol (Acharya et al., 2014). Subsequently, the resultant promoter fragment was sub-cloned into pUCPMAGUS and pKYLX71GUS to generate the pUPMUASCsV8CPGUS and pKMUASCsV8CPGUS plant expression vectors.

To generate the recombinant KP4 chimeric construct, the totiviral KP4 gene [UniProtKB-Q90121(KP4T_UMV4)] was fused with a 35-nucleotide untranslated region of AlMV RNA4 (5′ AMV; translational enhancer) and apoplast targeting sequence (aTP) of Arabidopsis 2S2 protein gene (Kroumova et al., 2013) at its 5′- end; and a hexa (6x) His-tag at its 3′- end. The above recombinant sequence was then cloned into the XhoI and SstI sites of the binary vector pKMUASCsV8CPGUS and pKCaMV35S²GUS (replacing GUS), to design the plasmids pKMUASCsV8CP-KP4His and CaMV35S²-KP4His. The pKMUASCsV8CP construct was also designed to serve as a vector control (VC).

The native and chimeric promoters along with CaMV35S and CaMV35S² (cloned in pUCPMAGUS) were electroporated into viable tobacco protoplasts following standard protocols (Acharya et al., 2014). Transient GUS activities from respective promoter constructs were calculated biochemically by fluorimetric GUS assay (Jefferson, 1987). For transient Agro-infiltration assays of the promoter constructs in tobacco, petunia, and tomato, Agrobacterium tumefaciens strain LBA4404 was transformed with respective promoter constructs following the freeze-thaw method as described previously (Chen et al., 1994). Leaves of whole plants of tobacco, petunia and tomato were mechanically infiltrated with individual Agrobacterium constructs (Yang et al., 2000). Quantitative fluorimetric GUS assay for each promoter construct was carried out 3–4 days’ post-infiltration (Jefferson, 1987; Ranjan et al., 2011; Acharya et al., 2014).

For comparative GUS activity analysis in agro-infiltrated leaves expressing CsVMV native deletion fragments cloned in pKYLXGUS, pKMUASCsV8CPGUS, pKMUAS35SCPGUS, pK35SGUS, and pK35S²GUS fragments; total leaf protein from transgenic seedlings (21 days old) were isolated and GUS activity analysis was performed according to standard protocols (Bradford, 1976; Jefferson, 1987). Histochemical GUS staining of 21 days old transgenic tobacco seedlings (T₂ generation) was performed according to a previously published protocol (Jefferson et al., 1987).

The native and chimeric promoter fragments along with KP4-His and VC constructs were used to transform tobacco plants by Agrobacterium-mediated gene transfer (Dey and Maiti, 1999) using LBA4404 strain. On an average, seventeen to eighteen T₀ transgenic tobacco lines were generated for each of the constructs. All transgenic plants showed normal growth and development. Subsequently, the T₁ generation seeds were subjected to segregation analysis (Patro et al., 2015). Transgenic lines with a chi-square value of <1.5 (p ≤ 0.05) were considered to be true transgenic lines. Independently re-generated transgenic lines showing proper segregation ratios (Kan^R:Kan^S::3:1) were selected and respective T₂ generation transgenic plants were propagated in greenhouse conditions [photoperiod: 16/8 h (light/dark), light intensity: 220 μmol/m²/s, temperature: 28 ± 2°C and humidity: 70–75%]. The transgenic tobacco plants expressing KP4-His along with VC lines were also compared for their agronomical parameters such as plant height, leaf architecture, seed fertility and flowering time. Likewise, the GUS activities of the transgenic plants expressing pKYLXGUS, pKMUASCsV8CPGUS, pKMUAS35SCPGUS, pK35SGUS, and pK35S²GUS was performed following the standard protocol (Bradford, 1976; Jefferson et al., 1987).

Total genomic DNA was extracted from 21-days old T₂ transgenic seedlings of individual lines expressing VC and pKMUASCsV8CP-KP4His, respectively (Dellaporta et al., 1983). Gene integration assays for uidA, KP4, rbcSE9 and nptII were performed in respective transgenic lines using gene-specific primer sets (Supplementary Table S1). Transgenic lines expressing native and chimeric promoter constructs along with KP4-His and VC plants were subjected to PCR amplification (of the genes mentioned above) following the standard protocol using Taq DNA Polymerase (Thermo Fischer Scientific; Cat No. #EP0402) having proofreading activity (Kumar et al., 2011) and all the above amplicons were sequenced by Sanger sequencing method employing a sequencer (Applied Biosystems 3500 Genetic Analyzer).

Briefly, 20 μg of genomic DNA isolated from selected T₁ progenies of transgenic and VC lines was digested using the XhoI restriction enzyme (Fermentas ER0691) overnight (16 h) at 37°C, electrophoresed on 0.8% agarose gel, blotted on nylon membrane (nylon-N+, Amersham) and finally probed with PCR amplified αP³²-labeled GUS and KP4-His probes using specific primers (Supplementary Table S1). The entire procedure of Southern Blotting was performed following a previously described protocol (Sambrook et al., 1989; Sarkar et al., 2015).

Total RNA from transgenic tobacco seedlings expressing the respective constructs were isolated using the RNeasy plant mini kit (Qiagen, Chatsworth, United States) and cDNA synthesis was performed. After that, Real-time PCR analysis was performed using Premix Ex TaqTM II (Perfect Real Time, Takara Bio Inc., Japan) employing the Opticon-2Real-time PCR (MJ Research, Bio-Rad; Model; CFD-3220). The transcript levels for individual constructs were calculated by the 2^-ΔΔC_T (CT-comparative threshold cycle) method (Livak and Schmittgen, 2001) and expressed as fold change in comparison to respective controls where 18S was used as the housekeeping gene (Kumar et al., 2012).

The uppermost fully expanded leaves of 6-week-old greenhouse grown plants (T₂ generation) of KP4L2#2, KP4L2#3, KP4L2#4, KP4L2#7, KP4L2#11, and VC were extracted and crushed in liquid nitrogen to make a fine powder. Next, the total soluble protein (TSP) was extracted from the crushed samples using an extraction buffer containing 1X PBS with 0.05% β-mercaptoethanol and plant protease inhibitor cocktail (Sigma- P9599-5mL), pH 7.0. An aliquot of 10 μg of total soluble protein obtained from transgenic lines was resolved on 15% SDS-PAGE for Western blot analysis. For detection of the recombinant proteins, primary antibody specific to the hexa- (6x) His-tag [(Mouse monoclonal His-probe (SC-57598) and Rabbit polyclonal His-probe (SC-803)] was used following the standard protocol (Sarkar et al., 2015).

KP4 was purified from 21-days-old transgenic KP4-His seedling (T₂) using Ni-NTA agarose (Qiagen) according to the manufacturer’s instructions with slight modifications as described earlier (Ma et al., 2005). Briefly, the seedlings of KP4L2#2 transgenic lines were homogenized (in 1X PBS, 1 mM phenylmethylsulfonyl fluoride and 1.5% PVP-40), filtered through layers of miracloth and centrifuged at 14,000 g for 20 min at 4°C. The supernatant obtained was dialyzed against 50 mM Tris-HCl (pH 7.0) buffer for 24 h at 4°C and adjusted to 40% ammonium sulfate saturation. After centrifugation at 30,000 g for 40 min, the enriched fraction obtained as pellet was solubilized, dialyzed (overnight) and filtered through a 0.45 μM membrane filter, and loaded on to a Ni-NTA agarose column. Finally, the column was washed with wash buffer (2 mM imidazole, 20 mM Na₂HPO₄, 100 mM NaCl). Ni-NTA agarose-bound KP4 was eluted with elution buffer (500 mM imidazole, 20 mM Na₂HPO₄, 250 mM NaCl). The eluted fractions were dialyzed extensively in 1x PBS and concentrated with a centrifugal filter device (Amicon 3 kDa cut-off).

The extraction of IF from transgenic leaves (T₂) expressing KP4 was performed following the infiltration- centrifugation technique (Chatterjee et al., 2017). The IF obtained post-extraction was enriched by 40% ammonium sulfate saturation and purified using Ni-NTA affinity purification. Finally, the eluted fractions were dialyzed and concentrated to detect KP4.

For agar-based killing zone assay, we followed a previously published protocol with slight modifications (Allen et al., 2011). The plant material for plate-diffusion assay was prepared by crushing 50 mg of leaf material in 1 ml of sterile 1X Phosphate buffered saline (PBS, pH 7.0). A. alternata (MTCC No.10833) and P. exigua var. exigua (MTCC No. 2321) cultures procured from MTCC (Microbial Type Culture Collection, Chandigarh, India) were grown on Potato Carrot Agar (PCA) medium (HiMedia- M696-500G) for 3 and 5 days, respectively. Fine wells were cut into the agar with the help of a cork borer (diameter = 0.5 cm) and 100 μl of the enriched test plant material prepared from both transgenic and VC lines were added to each well on either side of the fungal growth. Two other wells containing 100 μl each of sterile water and PBS were used as controls. The plates were then incubated at 25°C under constant light for A. alternata and, 12:12 h light:dark cycle for P. exigua var. exigua. The plates were photographed when clear zones of inhibition started to appear around the point of application of test samples.

To compare the protection efficacies of MUASCsV8CP and CaMV35S², we cloned the KP4-His downstream of the above promoters to generate pKMUASCsV8CP-KP4His and pKCaMV35S²-KP4His constructs. The recombinant constructs were then agro-infiltrated in healthy tobacco leaves. After 3–4 days of incubation, the infiltrated leaf segments were excised and total protein was isolated as described above. Equal concentrations of protein from both the constructs were used for the agar-based killing zone assays against A. alternata and P. exigua var. exigua.

Fungal resistance assays on detached leaves (employing A. alternata and P. exigua var. exigua) were performed according to a standard protocol with minor modifications (Emani et al., 2003). Briefly, young and healthy leaves were collected from 3 to 4 weeks old greenhouse grown plants and kept on wet paper towels in a petri-dish. On the other hand, A. alternata and P. exigua var. exigua were grown for 2 weeks on Potato Carrot Agar and Oat Meal Agar medium, respectively. Finally, an agar plug containing full mycelial growth was removed with the help of a cork borer (diameter = 0.5 cm) and agar plugs containing A. alternata and P. exigua var. exigua were placed on the adaxial surface of the leaves. In case of P. exigua var. exigua assay, the leaves were wounded with the help of a sterile needle at multiple points (6–7). Alongside, a mock leaf was kept to evaluate the effects of wounding inflicted by the needle. The petri-plates were sealed and kept in dark at 28°C. The leaves were photographed on the 14th and 18th day post-inoculation (dpi) of A. alternata and P. exigua var. exigua, respectively. The lesion area percentage was calculated with the help of millimeter graph paper method (Pandey and Singh, 2011).

The whole plant assays were performed following a previously published protocol. Healthy leaves of 8-weeks-old transgenic KP4L2#2, KP4L2#3, and VC plants were sprayed with conidial suspension of A.alternata and P. exigua var. exigua (Bithell and Stewart, 1998) using a sprayer. In case of P. exigua var. exigua, however, the leaves were wounded before applying the conidial suspension. The greenhouse conditions [photoperiod: 12:12 h (light:dark), light intensity: 220 μmol/m²/s, temperature: 25 ± 2°C and humidity: 90–95%] were kept extremely humid and the symptoms were recorded/photographed 20 dpi. The lesion area percentage was recorded as described above.

All experiment procedures were carried out in at least three independent replicates and the statistical analyses was performed using one-way analysis of variance (ANOVA) where a P-value of <0.05 was considered as significant.

The transient activities of each of the sixteen 5′ and 3′-end deletion constructs coupled to GUS reporter (pUCPMAGUS-CsVMV1 to pUCPMAGUS-CsVMVR3) (Figure 1A) were evaluated in tobacco protoplasts (cv. Xanthi Brad). Figure 1B depicts the average GUS activities obtained for each of the deletion constructs along with the CaMV35S promoter and an empty vector (EV) control with respective standard deviations (SDs). The data obtained clearly suggests that the relative GUS activity of the CsVMV8 (-215 to +166) was almost equivalent (∼1.1 times) to that obtained for the CaMV35S promoter; while the CsVMV7 (-256 to +166) ranked second. Furthermore, the TATA-less fragments CsVMVR2 (-565 to +16) and CsVMVR3 (-565 to -34) showed diminished GUS activities.

Additionally, we evaluated the transient activities of the above constructs in whole plants of Nicotiana tabacum Samsun NN by agro-infiltration assays. The average GUS activities along with respective SD were presented in Figure 1C. The data obtained was in accordance with that of the protoplast assays.

Finally, we raised transgenic tobacco plants by Agrobacterium mediated leaf disk transformation protocol as described in Methods. We successfully raised 7–10 independent transgenic lines expressing uidA gene under the control of CsVMV1, CsVMV7, CsVMV8, and CaMV35S promoter fragments, respectively. Next, we performed segregation and Southern-blot analysis of T₁ progeny transgenic plants. We observed, single copy integration of the transgene uidA in the transgenic lines (T₁ generation) CsVMV1L4, CsVMV7L7, CsVMV8L6, CsVMV8L13, CaMV35SL9, and CaMV35S²L11. Finally, from the Southern-blot analysis, those transgenic plants having single copy insertion of the transgene (uidA) and exhibiting appropriate segregation ratio (3:1), namely CsVMV1L4, CsVMV7L7, CsVMV8L13, and CaMV35SL9 were chosen for further analyses (Figure 2A).

We evaluated the stable GUS activities of 3-week old transgenic tobacco seedlings (T₂ generation). On analysis of the results, we observed that the CsVMV8 showed ∼1.1 times stronger activity than that of the CaMV35S promoter (Figure 2B). Additionally, we performed qRT-PCR analysis of the transgenic plants as described in section “Materials and Methods.” The relative accumulation levels of uidA-mRNA level in transgenic plants (T₂ generation) harboring the respective promoter constructs (viz. CsVMV1L4#2, CsVMV7L7#11, CsVMV8L13#7, and CaMV35SL9#3) confirmed ∼1.1-fold more expression of uidA under the control of CsVMV8 promoter in comparison to the CaMV35S promoter (Figure 2C). Finally, we obtained a fairly strong intensity of X-Gluc staining of 21 days-old transgenic seedlings expressing the CsVMV8 promoter (Figure 2D), in comparison to that obtained under other constructs.

Figure 3A represents the schematic map with essential components of the chimeric promoter MUASCsV8CP along with CaMV35S, CaMV35S² and MUAS35SCP. Upon transient activity analysis in tobacco protoplasts (Figure 3B) and in whole plants of tobacco, petunia, and tomato (Supplementary Figure S1), we detected that the GUS activity of MUASCsV8CP was ∼2.4 times stronger as compared to the CaMV35S promoter.

We raised transgenic plants harboring the MUASCsV8CP-GUS and CaMV35S²-GUS (a modified version of CaMV35S with double enhancer domain) (Kay et al., 1987) constructs individually as already described. Southern blot analysis was performed to determine the copy number of the uidA transgene in above plant lines (T₁ generation). The appearance of distinct southern-positive bands in MUASCsV8CPL4 and MUASCsV8CPL6 indicate integration of the transgene construct as a single copy insertion in the T₁ progeny plant genome (Figure 4A). The progenies (T₂ generation) of line MUASCsV8CPL6 exhibited expected segregation ratio and was chosen for further analyses.

We measured the GUS activities of the respective constructs using 21 days-old T₂ transgenic seedlings having the CaMV35S, CaMV35S², MUASCsV8CP, and MUAS35SCP promoters. The data obtained clearly showed that the chimeric promoter MUASCsV8CP, reported in this study has ∼2.4 and ∼1.1 times enhanced GUS activity as compared to CaMV35S and CaMV35S² promoters, respectively. The data also indicates toward the equivalence in activities of MUASCsV8CP and MUAS35SCP promoters (Figure 4B).

Additionally, we performed the Real-time PCR analysis and histochemical X-Gluc staining of the transgenic seedlings expressing GUS under the control of CaMV35S, CaMV35S², MUASCsV8CP, and MUAS35SCP promoters. The fold change as shown in Figure 4C indicates that the uidA-mRNA accumulation in MUASCsV8CP was nearly 1.8 times and 1.1 times as compared to CaMV35S and CaMV35S² promoters, respectively, while MUASCsV8CP and MUAS35SCP showed the previous trend of being equivalent. Finally, the histochemical staining also suggested strongest intensity using MUASCsV8CP and MUAS35SCP chimeric promoters (Figure 4D).

Using Agrobacterium-mediated gene transformation method we raised 18 independent T₁ generation transgenic tobacco plants (Kan^R) expressing totiviral KP4 driven by MUASCsV8CP. Figure 5A represents the schematic map of both KP4-His and VC constructs. We performed segregation analysis of T₁ generation transgenic plants as described in section “Materials and Methods.” Out of eighteen transgenic lines, the progenies (T₁ generation) of eight independent lines showed proper segregation ratio (Kan^R: Kan^S::3:1) and found to be Kanamycin (300 mg/L) resistant on half strength Murashige Skoog (MS) medium (Zhu et al., 1999) (Supplementary Table S2). Seeds from these selected plants (T₁ generation) showing proper segregation ratios were propagated to get homozygous T₂ transgenic plants.

The integration of transgenes was confirmed by PCR amplification using primers specific to KP4 (537 bp), nptII (822 bp), and rbcSE9 (646 bp) (Supplementary Table S1). We confirmed the integration of different components of KP4 expression construct in T₂ progenies of transgenic plant lines KP4L2#2, KP4L2#3, KP4L2#4, KP4L2#7, and KP4L2#11 whereas no amplification for KP4 was observed in the VC plant line (Figure 5B). We analyzed the sequence integrity of the amplicons and did not detect any mutations in the nucleotide sequences of KP4, nptII, and rbcSE9.

Next, Southern-blot analysis was performed with gDNA isolated from different KP4-His transgenic lines and a VC plant (T₁ generation progenies). Southern analysis confirmed the single copy insertion of KP4-His transgene construct in KP4L1, KP4L2, KP4L5, KP4L7, KP4L12, KP4L13, and KP4L15 transgenic tobacco plants (Figure 5C). KP4L4 however, showed two copies of the transgene. Further, the transcript analysis of KP4 in KP4L2 transgenic lines (selected on the basis of segregation analysis) was performed by Real-time PCR. The result obtained from independent transgenic lines KP4L2#2, KP4L2#3, KP4L2#4, KP4L2#7, and KP4L2#11 clearly showed the accumulation of KP4 in respective transgenic lines in the following order: KP4L2#2 > KP4L2#3 > KP4L2#7 > KP4L2#11 > KP4L2#4 as shown in Figure 5D.

In order to detect the KP4-His protein in transgenic plants, we isolated and concentrated TSP from individual transgenic seedlings (T₂ generation). After isolation and quantification of TSP, we performed western blot analysis using anti-His polyclonal (Figure 6A) primary antibody as described in section “Materials and Methods.” The KP4-His expressed in transgenic tobacco plants representing 2S2aTP-KP4-His was seen as a prominent band, the molecular mass being ∼18 kDa along with a few faint non-specific bands. To avoid the appearance of any contaminating bands, we performed western blot with anti-His monoclonal primary antibody (Figure 6B). In this case, a discrete single band of ∼18 kDa indicating the presence of 2S2aTP-KP4-His in TSP was confirmed.

To further detect the presence of KP4 in transgenic plants we performed nickel-nitrilotriacetic acid (Ni-NTA) affinity purification using TSP obtained from 21 days-old KP4L2#2 T₂ transgenic plants as described in section “Materials and Methods.” The C-terminal 6xHis tag present in MUASCsV8CP-KP4-His (plant expression construct) facilitated the purification of recombinant KP4 using Ni-NTA resin. The SDS-PAGE analysis clearly showed a band of ∼18 kDa after 40% ammonium sulfate enrichment coupled to Ni-NTA affinity chromatography process (Figure 6C). We also observed a prominent contaminating band of ∼55 kDa which we assume might be of the RuBiSCO large subunit.

In order to confirm the accumulation of KP4 in the IF of transgenic plants, we isolated and partially purified the intercellular fluid (as described in section “Materials and Methods”) from the transgenic line KP4L2#2. The presence of 2S2aTP from Arabidopsis 2S2 protein gene facilitated the accumulation of KP4 in the IF. The 40% ammonium sulfate enrichment and Ni-NTA affinity purification module was adopted for purifying KP4 from the IF of transgenic plants. Appearance of distinct bands of ∼18 kDa in the eluted fraction (as shown in Figure 6D) indicated that the KP4 is properly targeted to the plant apoplast or intercellular space. In this case also we observed the band of ∼55 kDa as a contaminant.

As KP4 blocks voltage-gated calcium channels of fungi, we sought to check the efficacy of plant-derived KP4 against two deuteromycetous fungal phyto-pathogens: A. alternata and P. exigua var. exigua using the ‘plate-diffusion’ assay (Allen et al., 2011). Enriched protein extracts for KP4L2#2, KP4L2#3, and KP4L2#7 were evaluated for anti-fungal activity as shown in Figures 7A–C (A. alternata) and Figures 7D–F (P. exigua var. exigua). It was evident that the KP4 activity represents a clear zone of inhibition around the point of application. This inhibition might be due to the blockage in calcium uptake by the fungal cells causing precocious hyphal disintegration.

Using KP4 as the gene of interest, we checked the efficacies of the MUASCsV8CP and CaMV35S² promoters in the frame of fungal inhibition against A. alternata and P. exigua var. exigua. For this assay, we used transiently expressed KP4 from tobacco leaves infiltrated with pKMUASCsV8CP-KP4-His and pKCaMV35S²-KP4-His constructs (Supplementary Figure S3). Although we observed a slightly enhanced inhibition in case of pKMUASCsV8CP-KP4-His, we assume equivalence in activities of both promoter constructs.

Deuteromycetous necrotrophic fungal pathogen Alternaria and hemi-biotrophic pycnidial pathogen Phoma causes foliar diseases in a wide variety of vegetables and annual plants (Momol et al., 2004; Mamgain et al., 2013). A. alternata causes brown spot disease on tobacco which is characterized by the appearance of roughly circular necrotic spots with concentric rings surrounded by yellow halo. On the other-hand, P. exigua var. exigua causes Phoma blight or ragged leaf spot in tobacco which is characterized by the appearance of irregular brown to ashy gray spots. In order to evaluate the anti-fungal efficacy of transgenic plants expressing KP4, both A. alternata and P. exigua var. exigua were used as test pathogens. Prior to performing the bio-assays with the above fungal strains (procured from MTCC), we checked for their virulence toward infecting tobacco plants. We found that both the pathogens could successfully infect mature tobacco leaves and develop visible symptoms (data not shown).

The disease resistance assays for both the pathogens were carried out with detached leaves kept on wet paper towels on petri dishes as described in section “Materials and Methods.” The detached-leaf technique is a routinely used method to determine the disease resistance of a particular transgenic plant against necrotrophic pathogens (Emani et al., 2003). In case of A. alternata, yellowish halos began to appear 7–8 days dpi while in P. exigua var. exigua, irregular brown spots appeared 10–11 dpi. The lesion area was measured 2 weeks post-inoculation for A. alternata (Figures 8A,B) while 18 dpi for P. exigua var. exigua (Figures 9A,B). The results revealed a high degree of protection in transgenic plants expressing KP4 as compared to the VC, as supported by the observation that the leaves of transgenic plants were found to be unaffected or developed smaller lesions than the VC plants.

Following detached leaf assay, the lesion area percentage was determined using ‘Millimeter graph paper method’ as described in section “Materials and Methods.” We found, lesser lesion area in case of KP4 transgenic leaves as compared to the control plants (Figures 8C, 9C). These results suggested an enhanced anti-fungal activity of KP4 against these foliar fungal pathogens, which can be considered as significant.

As described in section “Materials and Methods,” three independent experiments were carried out with whole plants of transgenic and VC lines. In all sets of experiments, the VC leaves developed distinct lesions much earlier than the KP4 transgenic plants. Significant lesions of A. alternata infection were observed 9–10 dpi in VC plants (Figure 10A). On an average, we observed that the VC plants showed 16.4% lesion area, while KP4L2#2 and KP4L2#3 showed 5.5 and 5.9% lesion areas, respectively (Figure 10B). Likewise, we observed higher degree of lesions caused by P. exigua var. exigua in VC plants as compared to the KP4 transgenic lines KP4L2#2 and KP4L2#3 (Figure 11A). We observed 12.4% lesion area (as a mean value) in VC plants, in comparison to 6.7 and 5.7% lesion areas in KP4L2#2 and KP4L2#3 transgenic lines, respectively (Figure 11B).

Consistent with our results of detached-leaf assays for A. alternata and P. exigua var. exigua, we found significantly lesser leaf-area exhibiting symptoms in case of KP4 transgenic leaves as compared to the VC plants. Although, some of the leaves showed inconsistent results, the percent lesion area clearly suggested inhibition and/or delayed lesion development in transgenic plants expressing KP4. The inhibition found in detached-leaf and whole plant assays further proved the anti-fungal efficacy of KP4 against both A. alternata and P. exigua var. exigua.