Plant transformation and characterization

Binary vectors containing either the C. elegans gene Ced9 under the control of a maize Ubi-P and a cauliflower mosaic virus 35s terminator (35S-T)8 or the M. acuminata ssp. malaccensis RGA2 under the control of the Agrobacterium tumefaciens Nos-P and terminator sequences17 were used in this study M. acuminata Cavendish cv. GN (AAA subgroup) embryogenic cell suspensions were prepared from immature male flowers and transformed using the centrifugation assisted A. tumefaciens-mediated method18. Following selection, plants derived from single embryos were regenerated and were screened for the presence of the respective transgene by PCR using specific primers. A total of six RGA2 and nine Ced9 lines were generated, and up to 10 replicates of each transgenic line was multiplied in tissue culture for field analysis. Both the number of transgenic lines and the number of replicates per line permitted in the field trial was limited by the licence conditions (DIR 107) imposed by the Office of the Gene Technology Regulator (OGTR). Plants derived from non-transformed cell suspensions were also generated as controls. Before field planting, tissue-culture plants were acclimatized in a secure shade house over a 3-month period by which time they had reached a height of ~35 cm.

Field trial design

The field trial was conducted on a commercial banana plantation site located at Lambells Lagoon, Northern Territory, Australia. The site had previously been used to grow Cavendish banana plants and has a history of high incidence of TR4 infection. The field trial comprised two plantings, the first in January 2012 and the second in May 2012. Owing to the seriousness of the threat of TR4 to the Australian banana industry, it took 8 years from the initial generation of the transgenic lines to identify a suitable field trial location and obtain permission from the plantation owner, the biosecurity regulators in both Queensland and the Northern Territory, and the OGTR to conduct this trial. Unfortunately, the trial was only conducted for 3 years, not the intended 5 years, because of a forced quarantine termination order due to another disease.

Transgenic lines were planted in a randomized design, with rows containing blocks of ten transgenic plants and each block separated by four non-transgenic control plants. The control plants included cell line control plants (cv Cavendish selections GN and/or Williams) from QUT in addition to tissue culture plants of Williams, GCTCV 218 and DPM25 supplied by Mission Beach Tissue Culture Nursery, Queensland. To increase the level and uniformity of inoculum pressure, a small segment of pseudostem taken from TR4-infected banana plants growing outside the trial site was buried between each plant.

When a banana is planted, the plant crop, a pseudostem comprising the petioles of leaves grows from the basal corm with the vegetative meristem remaining at the base of the pseudostem. When flowering is initiated, the meristem is pushed up through the center of the pseudostem, the fruit bunch emerges through the top of the pseudostem and develops through to maturity. After the bunch is harvested, this initial pseudostem dies back and another new pseudostem, known as the first ratoon, grows from a different meristem on the corm. This process can be repeated indefinitely. The trial consisted of the plant crop and up to three ratoon crops.

Assessment of symptoms and bunch size

Plants were regularly assessed over three crop cycles (~3 years) for the development of characteristic external symptoms of Fusarium wilt disease1. The pseudostem of plants exhibiting typical external symptoms was inspected to confirm the presence of characteristic internal reddish-brown vascular discoloration associated with infection by Foc. If a plant shows the characteristic external symptoms of wilting and/or yellowing of their leaves, it almost invariably develops severe vascular necrosis followed by death of that pseudostem. In some instances, the symptomatic pseudostem would die but an apparently healthy ratoon pseudostem would grow from the basal corm. The first observation of symptoms was recorded for each plant; however, ratoons from diseased plants were allowed to develop naturally until the completion of the trial period. At the completion of the trial period, all plants were inspected for external and internal symptoms. The presence of the internal vascular discoloration, which is highly characteristic of Foc TR4 infection, was assessed in all remaining plants by cutting the pseudostem of all plants ~0.5 metres above ground level. Where vascular discoloration was observed, a pseudostem sample of ~3 cm × 1 cm was taken from the leading edge of the discoloration. Where no vascular discoloration was observed, a similar sample was taken from an equivalent area. Pseudostem samples from 118 plants were collected and were sent by air freight to Wageningen UR, Plant Sciences Group, The Netherlands, for confirmatory diagnosis of Foc infection.

To avoid any bias, all plants in the trial were assigned a unique Field Trial Number which contained no identifying information. Further, the assessment of disease symptoms during the 3 year trial period was done independently by the farm manager (MS) who has more than 20 years experience managing a TR4-infested commercial banana farm. At the completion of the trial, assessment for internal and external symptoms was done by two of the authors (JD and RH).

At various times during the trial, the size of mature fruit bunches on healthy transgenic and non-transgenic plants was visually assessed and rated into three categories, <6, 6–8, or >8 hands per bunch.

Diagnosis of Foc infection

The pseudostem samples were used for a combination of fungal isolation and molecular diagnostic analyses by PCR13,14 and quantitative PCR (qPCR kit: “Foc TR4 DNA identification by Real-Time PCR, Clear Detections”, The Netherlands). For DNA isolation from plant samples, small pieces of pseudostem tissue were selected and subsequently lyophilized in an Epsilon 1-4/2–4 LSC plus freeze dryer (Martin Christ) for 3 days. For a subset of 49 samples that were collected from the above mentioned materials, 2–4 pieces of the same tissue were sterilized with 1% hypochlorite, rinsed with Milli-Q water, dried in filter paper, and placed on Komada medium for fungal re-isolation13. After 5 days, emerging Foc colonies were sampled for PCR analysis13, as well as qPCR. Total plant and fungal DNA was extracted using the Sbeadex maxi plant Kit (LGC Genomics). DNA extractions from all plant and fungal samples were independently repeated at least twice. Analytical PCRs were also technically repeated twice. qPCR was conducted in a total volume of 20 µL reaction mixture containing 10 µL of Clear Detections PCR mix, 2 µL of Foc TR4 primer set, 3 µL of PCR enhancer, 0.2 µL of ROX reference dye II (Takara), 3 µL of DNA template, and Milli-Q water in a 7500 Real-Time PCR system (Applied Biosystems). Thermal cycling conditions for amplification were an initial enzyme activation at 95 °C for 3 min, followed by 35 cycles each consisting of 95 °C for 10 s, 63 °C for 60 s, and 72 °C for 30 s. Finally, for the PCR melt curve 0.2–0.5 °C, steps at 72–95 °C were included. The amplification results were analyzed with 7500 Real-Time PCR software v 2.3 (Applied Biosystems).

Transgene expression analysis

Owing to quarantine restrictions, we were unable to transport samples from banana plants within Australia for analysis. Therefore, transcript analyzes were done using samples taken from the original mother plants stored in tissue culture at QUT, Brisbane, Australia. Tissue culture plants of wild-type (WT) TR4-susceptible and resistant M. acuminata ssp. malaccensis were used as controls19,20.

Total RNA was extracted from 200 mg of root tissue using a protocol21 that was modified by increasing the tissue:extraction buffer ratio to 8, including a centrifugation step (18,000xg for 5 min) before the initial solvent extraction and the omission of phenol from all extraction steps. RNA (3 μg) was DNase-treated using an RQ1 RNase-free DNase Kit (Promega) and DNA-free RNA samples (1.8 μg) were reverse-transcribed to complementary DNA using an oligo(dT)18 primer and the GoScript Reverse Transcription System (Promega) according to the manufacturer’s instructions. Subsequently, cDNA samples were diluted either 1:10 (v/v) or 1:8 (v/v) in RNase-free water before use in RT-PCR and qRT-PCR, respectively. To ensure complete removal of potential gDNA contamination in our samples before RT-PCR and qPCR, total RNA, DNase-treated RNA, as well as cDNA was tested by PCR using the cyclophilin (CYP) housekeeping gene primers (Supplementary Table 3).

Reaction mixes for RT-PCR contained 1 × GoTaq Green master mix (Promega), 0.25 µM of each primer (Supplementary Table 3), diluted cDNA (2 µL), and nuclease-free water in a final volume of 20 µL. Thermal cycling conditions included a 2 min denaturation step at 94 °C followed by 35 cycles of 94 °C for 20 s, 55–62 °C (primer dependent) for 30 s, and 72 °C for 1 min Kbp-1 of expected amplicon size, and a final extension at 72 °C for 5 min.

qRT-PCR was performed on a CFX384 Touch Real-Time PCR Detection System (Bio-Rad) using the SYBR Green I technology. Per 10 μL reactions, 2.5 μL of diluted cDNA was added to 1 × GoTaq qPCR Master Mix (Promega) premixed with primers (Supplementary Table 3) at a final concentration of 0.2 μM. The following amplification program was used: Hot-Start polymerase activation at 95 °C for 2 min, followed by 45 cycles of 10 s denaturation at 95 °C and 30 s annealing/extension at 60 °C. At the end of each run, a dissociation curve was produced from 65–95 °C, to confirm the specificity of the amplicon from each primer set. A standard curve of eight serial two-fold dilutions of cDNA was used to determine the qPCR efficiency of each of the primer sets used22. All PCR reactions displayed a correlation coefficient R 2>0.98 and efficiencies >99% (Supplementary Fig. 2). All samples were analysed in triplicate and each run included triplicate non-template control reactions for each of the primer sets used on that run. In addition, selected samples from each run were electrophoresed through 2 % agarose gels to validate production of a single amplicon.

Relative expression levels were calculated using CFX Manager 3.1 (Bio-Rad) software and the ΔΔCT method23. Ct data obtained from target gene of interest were normalized using Ct values from the two stable reference genes CYP and ribosomal protein S2 (RPS2), and expressed relative to the values of line RGA2-3. All primers were designed using the Primer3Plus freeware (http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi).

Southern blot analysis

For determination of transgene copy number integration by Southern analysis, total nucleic acid was extracted24 from banana leaf tissue, treated with RNAse A, and 15 μg aliquots of genomic DNA were digested overnight with 20 U of restriction enzyme HindIII or XmaI (New England Biolabs) overnight at 37 °C. Digested DNA was electrophoresed through 0.9% agarose gels, transferred to a nylon membrane (Roche), and UV cross-linked. Gene-specific probes were amplified by PCR using Taq DNA polymerase (Sigma-Aldrich) in reaction mixes containing the appropriate primers (Supplementary Table 3), 2 ng of plasmid template and DIG PCR labeling mix (Roche). Hybridization of the probe was done under standard conditions25 and detection was achieved using CDP-star (Roche), as per the manufacturer’s instructions. X-ray films (Fuji) were exposed for up to 1 h depending on signal intensity and developed manually.

Isolation and analysis of endogenous Cavendish RGA2 homologs

The entire open reading frame of the endogenous Cavendish RGA2 sequence was amplified from total nucleic acid prepared24 from leaf tissue collected from WT GN plants. Primers RGA2geneF (5′-ATGGCTGGTGTCACATCACAGGCAG-3′) and RGA2geneR (5′-TCAGGTGGTGCTACAGCGACATGG-3′) were designed based on the M. acuminata ssp. malaccensis RGA2 sequence9. PCR was carried out using either GoTaq Long Master Mix (Promega) or Expand Hi-Fidelity Enzyme Mix (Roche). GoTaq Long PCR mixtures contained 20 μL 2× GoTaq Long Master Mix, 10 ρmol of each primer, 0.5 μL TNA extract, and 17.5 μL water. Cycling conditions were 95 °C for 2 min, followed by 35 cycles of 95 °C for 15 s, 50 °C for 15 s, 65 °C for 8 min, and a final extension at 72 °C for 10 min. PCR using Expand was carried out according to the manufacturer’s instructions, with cycling as above except for extension carried out at 68 °C. PCR products were analyzed on 1% agarose gels and visualized using SYBR safe DNA gel stain (Thermo Fisher Scientific). PCR products of the expected size were excised from gels, ligated into pGemT Easy (Promega), and heat-shock transformed into competent Escherichia coli XL-1 Blue cells (Invitrogen). Blue/white selection was used to identify putative recombinant colonies, with six clones derived from each PCR amplification selected and grown in overnight cultures. Plasmid DNA was purified using the Wizard Plus SV Miniprep DNA purification system (Promega) and digested using NotI to identify clones with inserts of the expected size. Plasmids were then sequenced using the BigDye Terminator mix v3.1 (Applied Biosystems) according to the manufacturer’s instructions, with sequence reads generated at the Queensland University of Technology Molecular Genetics Research Facility. Specific primers were used to completely sequence the inserts in both directions. Raw sequence reads were compiled into full-length sequences using the VectorNTI Advance V11 software (Life Technologies) program ContigExpress, whereas sequence alignments were carried out using AlignX. Consensus sequences of the WT GN RGA2 homologs were compared with the M. acuminata spp. malaccensis RGA2 sequence, to determine the level of nucleotide and amino acid sequence similarity.

Statistical analysis

Differences in total proportions of infected plants between the treatment and control groups, and between lines, were assessed using single and mixed effects generalized linear models and corresponding analyses of variance, assuming a binomial response and with the respective addition or subtraction of 0.5 for 0 or total infections.

Pairwise comparisons between treatments and lines, adjusted for multiple comparisons, were evaluated using Tukey's HSD test. Model fit was assessed using a χ 2-test of deviance and the Akaike information criterion (AIC).

The equivalence of ratings of fruit bunches from control, RGA2 lines and the Ced9 lines as number of hands in the bunch (<6, 6–8, and >8) was assessed using a χ 2-test of homogeneity.

The hypothesis of a (linear) relationship between the proportion of infections and the expression level for each of the RGA lines and the GN control line was assessed using parametric (Pearson) and nonparametric (Spearman) correlations, and the associated tests.

Statistical significance was asserted at the 5% level (p < 0.05). The analyses were undertaken in R using base statistical functions and the packages lme4, lmer, and multcomp.

Data availability

The authors declare that all data supporting the findings of this study are available within the article and/or its Supplementary Information file. All relevant data are also available from the authors upon request.