The BcSnod1 and BcSnod2 genes (the coding region downstream of the signal sequence) were successfully amplified from B. cinerea B05.10 genomic DNA. Fragments of approximately 370 and 410 bp correlated with the expected sizes from genomic sequencing indicating the absence of introns. BcSnod1 and BcSnod2 expressed well in bacteria as fusion proteins with N-terminal hexahistidine tags. Both were insoluble in Luria broth and minimal M9 media, even at reduced growth temperature of 16 °C, see Figure 1. Insoluble protein was denatured using 8 M urea and refolded by dialysis reduction of the urea concentration. Both BcSnod fusion proteins were soluble in the final dialysis buffer of 20 mM Tris, pH 6.8. Approximate molecular weights were as expected, with measured values of 15 kDa for BcSnod1 and 15.5 kDa for BcSnod2 correlating well with calculated weights of 14.70 kDa and 15.20 kDa. Refolded yields for both were approximately 2 milligrams per liter of culture.

In planta transient assays were conducted by injecting leaves with refolded, bacterially produced BcSnods. Young tomato leaves were injected with 100 μL of BcSnod protein (2 mg/ml in 20 mM Tris, pH 6.8). The area injected with either BcSnod started to discolor 3 days post inoculation (dpi), see Figure 2. Six attempts were made for each protein in which four BcSnod1 injections produced discoloration while three BcSnod2 injections produced discoloration. In this case, BcSnod induced discoloration did not turn into necrosis. Due to poor solubility of the bacterially expressed protein, injections of more concentrated BcSnod protein were not pursued. Controls of empty vector transformed bacteria, treated identically as the BcSnod samples in the same final buffer of 20 mM Tris, pH 6.8, did not cause discoloration.

Pichia pastoris transformation of BcSnod1 and BcSnod2 in the 10 kb pPic9k vector construct yielded approximately 3.0 × 10⁴ colonies/μg of DNA. Five transformants for each were screened using PCR amplification. Only a single band of expected size was observed, indicating transformation by both BcSnod genes. BcSnod1 expressed well as a secreted protein, yielding greater than ten of milligrams per liter of starting culture in minimal glycerol media induced in 100 mL of minimal methanol media. After HPLC purification, the total amount of BcSnod1 obtained was 6 mg (see Figure 3). In contrast, BcSnod2 did not express in P. pastoris in any of the 5 clones tested. PCR amplification re-confirmed the presence of BcSnod2 gene in the genome but no protein was ever detected in the culture media. No sign of BcSnod2 overexpression was found intracellularly either. As similar as the sequences are, BcSnod1 and BcSnod2 certainly behave differently in yeast overexpression. A control strain of Pichia pastoris transformed with an empty pPic9k was also constructed. Treated, induced, and prepared identically, the same HPLC fractions from the yeast vector only served as a background control.

When injected in tomato (Solanum lycopersicum) leaves, BcSnod1 obtained from P. pastoris initiated discoloration in the area of infiltration within 3 days. Discoloration turned into necrosis around 10 days after infiltration and spread to adjoining areas within a few days. Neither the leaf veins nor midvein prevented the propagation of necrosis, which continued through the duration of the experiment (i.e., to Day 30), see Figure 4. Control extracts from P. pastoris with the empty pPic9k vector induced yellowing in the region of injection after 10 dpi, but discoloration did not spread to adjoining regions as seen for BcSnod1. Even after 30 days minimal necrosis is observed, all centered at the site of infiltration. The same infiltration assay was performed in tobacco (Nicotiana tabacum var. Xanthi) leaves. For tobacco, the area of injection showed discoloration (an observation identical to the hypersensitive response as in Frías et al. [8]) within a few days after infiltration only against BcSnod1. Likewise, necrosis was readily observable at day 15 again only for BcSnod1 infiltrated leaves. Unlike tomato, necrosis in tobacco was limited to the area of injection and did not cross leaf veins and spread to other areas, see Figure 5.

Two types of detached leaf assays were performed, one with infiltration of protein into the leaves while the other tested surface exposure by pipetting a drop of protein solution onto the leaves. Healthy tomato leaves were cut off 4 week old plants and placed in a moist box in environmental chamber. For infiltrated leaves, 130 μg of protein solution was injected. Hypersensitive response was observed starting the first day after infiltration for BcSnod1 but not the control, see Figure 6. For BcSnod1, discoloration of the area of infiltration was observed after 3 days with maximum discoloration around day 10. Discoloration did spread to other areas. For the control, some discoloration was observed after 5 days but of very low intensity compared to BcSnod1. Discoloration for the control did not spread.

For surface exposure in detached tomato leaves, a 10 μL drop of solution containing 7 µg of BcSnod1 was placed on the upper side of the leaf. Signs of hypersensitive response were observed within 3 days, and necrosis identified 7 days post-infiltration, see Figure 7. No discoloration, much less necrosis, was observed for an equivalent volume of vector only control pipetted onto the leaf surface. The study was terminated at 8 days due to demise of the detached leaves. These findings show that BcSnod1 is able to elicit necrosis simply by exposure to the plant cell surface. Infiltration significantly expedited the process, but it appears that BcSnod1 (or at least some part of it) is able to enter plant cells and initiate cell death.

Both recombinant BcSnod1 and BcSnod2 displayed phytotoxic activity. However, the activity of bacterially produced BcSnod1 protein was more variable than Pichia produced protein. Noticeable differences were observed for the plant hosts tomato and tobacco in response to BcSnod1. In tomato, the area where the protein was infiltrated initially got discolored and then necrotized. Necrosis started spreading to regions adjoining the region of infiltration. This was different in tobacco plant in which the necrosis could only be observed in the area of infiltration and did not spread to adjoining regions.

BcSnod1 and BcSnod2 genes were amplified from genomic B. cinerea B05.10 DNA. The primers used for BcSnod1 were 5'-GTGCATATGATCACCGTCTCCTACG and 5'-GTGCTCGAGTTACAATCCACAAGCACTCTTGTCG. For BcSnod2, the primers used were 5'-GTGCATATGATCCAAGTAACCTACG and 5'-GTGCTCGAGCTAAGCCTTACACGGTGATCC. PCR products for both genes were first cloned into pGemTEasy (Promega Corporation, Madison, WI). For subsequent recombinant bacterial expression, BcSnod genes were excised using Nde1 and Xho1 restriction sites and ligated into pET28b (Novagen/EMD/Merck KGaA, Darmstadt, Germany). For yeast expression in Pichia pastoris, excised genes were inserted into pPic9k (Invitrogen/Life Technologies, Grand Island, NY) using EcoR1 and Not1 restriction sites. DNA sequencing confirmed the integrity of all clones.

For bacterial expression of both BcSnod1 and BcSnod2, chemically competent Rosetta E. coli were transformed with kanamycin resistant pET28b expression vectors containing the BcSnod protein of interest. Protein expression in Luria broth was induced at an OD₆₀₀ of 0.8 by adding 1.0 mM isopropyl β-D-thiogalactopyranoside and continued incubation at 37 °C for 4 h. Cells were harvested by centrifugation and stored at −80 °C. After lysis by sonication, insoluble BcSnod protein was collected by centrifugation at 20 kg for 20 min at 0 °C. The pellet, containing BcSnod, was then denatured in 8 M urea with 20 mM Tris pH 7.0, 250 mM NaCl and 3 mM DTT for refolding. Using step-wise dialysis, the protein was refolded by urea removal. Soluble BcSnod was further purified by metal chelation chromatography, eluted with 150 mM imidazole. The final dialysis buffer was 20 mM Tris pH 7.0 and 3 mM DTT.

For expression in Pichia pastoris (GS115 His⁻, ATCC-20864), the BcSnod vector of interest was linearized with BglII and Pichia transformed via electroporation. For electroporation, 80 µL of elctrocompetent cells were mixed with 3 ng of DNA and a 1.5 kV, 25 μF, 200 Ω electroporating pulse was applied. Screening for presence of BcSnod insert was carried out by PCR using the AOX primers. Mut^S mutants of transformed Pichia were used for recombinant expression. For screening, single colonies were inoculated in minimal glycerol media and grown until the OD₆₀₀ reached 2.0. The cells were pelleted and resuspended in minimal methanol media for protein expression. Every 12 h, 0.5% methanol was added to the media to maintain expression of protein.

For large scale production, a colony of Pichia expressing BcSnod1 was grown overnight in 1 liter of minimal glycerol media, spun down, and then resuspended in 100 mL of minimal methanol media. Every 12 h, 0.5% methanol was added for protein expression. After 10 days, the supernatant was collected and concentrated to a final volume of 5 mL. The concentrated solution was applied to a C18 HPLC column for purification. A gradient of acetonitrile in 0.05% trifluoroacetic acid was used to purify BcSnod, which eluted at approximately 55% acetonitrile.

Two month old tomato (Solanum lycopersicum) and four month old tobacco (Nicotiana tabacum var. Xanthi) plants were used for the transient activity assays. All plants were grown in an environmental chamber (24 °C, 14 h, 40W/m² light intensity, 10 h dark). For detached leaf assays, leaves were cut from the plant and kept in a wet box in the environmental chamber. For the transient assay with bacterially expressed protein, 200 μg of BcSnod1 or BcSnod2 in 100 μL were infiltrated in the midvein of tomato leaves using a syringe and 30.5 gauge needle. For the transient assay with Pichia expressed protein, 15 μg of BcSnod1 protein in 100 μL was infiltrated in the midvein of tomato and tobacco leaves using a syringe and 30.5 gauge needle. In the detached leaf assays, leaves were infiltrated with 100 μL containing 130 μg of protein after plugging out of the plant. In the surface exposed detached leaf assay, 10 µL of 7 μg protein was pipetted on he leaves and kept in wet box in environmental chamber. As a control, extracts from Pichia with no insert were infiltrated or pipetted on the leaves.