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Resistencia A Tr4
Resistencia A Tr4
Banana (Musa spp.) is a staple food for more than 400 million people. Over 40% of world
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production and virtually all the export trade is based on Cavendish banana. However,
Cavendish banana is under threat from a virulent fungus, Fusarium oxysporum f. sp. cubense
tropical race 4 (TR4) for which no acceptable resistant replacement has been identified. Here
we report the identification of transgenic Cavendish with resistance to TR4. In our 3-year field
trial, two lines of transgenic Cavendish, one transformed with RGA2, a gene isolated from a
TR4-resistant diploid banana, and the other with a nematode-derived gene, Ced9, remain
disease free. Transgene expression in the RGA2 lines is strongly correlated with resistance.
Endogenous RGA2 homologs are also present in Cavendish but are expressed tenfold lower
than that in our most resistant transgenic line. The expression of these homologs can
potentially be elevated through gene editing, to provide non-transgenic resistance.
1 Centre for Tropical Crops and Biocommodities, Queensland University of Technology, Brisbane, 4001 Queensland, Australia. 2 Darwin Banana Farming
Company Lambells Lagoon 0822 Northern Territory Australia. 3 Wageningen University and Research Centre, Plant Research International, Wageningen,
6700, The Netherlands. 4 Science and Engineering Faculty, Queensland University of Technology, Brisbane, 4001 Queensland, Australia. 5Present address:
Sugar Research Australia Indooroopilly 4068 Queensland Australia. 6Present address: Unidad de Biotecnologia Centro de Investigacion Cientifica de Yucatan
Merida 97205 Yucatan Mexico. Correspondence and requests for materials should be addressed to J.D. (email: [email protected])
F
usarium wilt or Panama disease is a devastating disease of under the control of the maize polyubiquitin promoter (Ubi-P)
bananas. In the first half of last century, it caused one of the and RGA2 was under the control of the Agrobacterium nopaline
most serious plant disease epidemics in history. During that synthase promoter (Nos-P). These cassettes were transformed
period, F. oxysporum f. sp. cubense (Foc), the fungus responsible separately into the Cavendish cultivar Grand Nain (GN). After
for Fusarium wilt, caused a major epidemic in commercial screening primary transformants by PCR, five RGA2 lines
banana plantations in South and Central America in the then (RGA2-2, 3, 4, 5, and 7) and five Ced9 lines (Ced9-17, 19, 21, 22,
dominant export cultivar Gros Michel1. This epidemic was caused and 26), together with untransformed controls, were selected for
by Foc race 1 and led to the almost complete replacement of Gros Southern analysis. Each of the RGA2 lines contained multiple
Michel with Cavendish, which is resistant to Foc race 1. transgene copies in addition to three endogenous RGA2 homo-
Cavendish now accounts for >40% of world's banana production logs present in untransformed control plants (Fig. 1c). The five
and completely dominates the banana export market, which Ced9 lines contained between one and many transgene copies
amounts to 15% of world production. Despite this, Foc race 1 with no endogenous homologs identified (Fig. 1d).
continues to cause significant disease in a wide range of other
locally produced and traded banana cultivars2. Foc invades
through the roots before entering the corm and pseudostem Field assessment of transgenic banana plants. To determine
where it causes extensive necrosis leading to plant death3. The whether these transgenes could confer TR4 resistance, we asses-
fungus is disseminated in infested soil, infected planting material sed the ten independent transgenic lines (RGA2-2, 3, 4, 5, and 7;
and water including irrigation water and floods, and can remain Ced9-17, 19, 21, 22, and 26), along with five additional PCR-
in the soil for >40 years1. In the early 1990s, another form of Foc, positive transgenic lines but for which Southern analysis had not
tropical race 4 (TR4) was recognized in South East Asia4, which been done (RGA2-6; Ced9-10, 15, 23, and 31), for resistance in a
differed from Foc race 1 in that it infects and kills Cavendish as field trial over a 3-year period. The trial site was a commercial
well as a number of other important race 1-resistant cultivars. Foc banana plantation in the Northern Territory of Australia where
TR4 now devastates Cavendish plantations in Indonesia, Malay- TR4 has become endemic and where Cavendish banana plants
sia, China, the Philippines, Australia, and Mozambique. It con- had previously been devastated by the disease. Non-transgenic
tinues to move internationally with recent reports of its spread controls included in the trial were the TR4-susceptible Cavendish
into Jordan, Pakistan, and Lebanon4,5, and it is highly likely that cultivars GN and Williams, in addition to cultivars GCTCV 218
the fungus and the disease will continue to spread particularly in and DPM25 (Dwarf Parfitt Mutant). GCTCV 218 is a variant of
south and south east Asia. Of the banana-producing continents, Giant Cavendish selected in Taiwan for its tolerance to TR46,
only the Americas have yet to record TR4. The disease now poses whereas DPM25 is a ɣ-irradiated selection of Cavendish cultivar
a very significant threat to commercial banana production Dwarf Parfitt, which has resistance to another race of Foc, sub-
worldwide and, together with race 1, severely limits the number tropical race 4 (STR4)12. The trial location has a tropical climate
of banana cultivars suitable for either large scale or smallholder with about 90% of its annual rain usually falling during the wet
production. season (November–April). The trial was planted during the wet
There is no effective chemical control for TR44 and efforts to season in early 2012, according to a randomized design, and run
contain the disease through inter- or intra-national quarantine for 3 years, as banana is a perennial crop. To increase the
have clearly been ineffective as evidenced by the continued spread inoculum pressure, infected plant material was buried between
between continents, countries, and regions. Locally, efforts such each plant.
as destruction of infected plants, isolation of infested areas, and The trial was regularly inspected for plants showing typical
disinfection of vehicles, machinery, and clothing are likely, at TR4 symptoms such as wilting and/or leaf yellowing (Fig. 2a).
best, to slow the rate of spread. Although somaclonal variants of The pseudostems of symptomatic individuals were further
Giant Cavendish (Giant Cavendish Tissue Culture Variants examined for the presence of the reddish-brown vascular
(GCTCVs)) with varying levels of tolerance to TR4 have been discoloration characteristic of TR4 infection (Fig. 2b). The disease
generated in Taiwan through tissue culturing6, these are con- status of the plants, based on this visual assessment, was recorded
sidered a short-term solution to disease control at best due to lack at regular intervals in both the wet and dry (May–October)
of immunity and undesirable agronomic traits7. The lack of seasons (Figs. 3a,b and Table 1). For the final assessment, the
effective TR4 control measures and the devastating impact of the pseudostems from all of the surviving plants were scored for
disease make the deployment of resistance genes an obvious and vascular discoloration and a selection of samples taken and tested
attractive strategy. Here we report the generation and field- for TR4 by a combination of fungal isolation and PCR-based
trialling of transgenic Cavendish banana plants and the identifi- assays13,14. Of the 118 samples, 107 were from plants showing no
cation of lines with robust resistance to TR4. vascular discoloration and all tested negative for TR4. The 11
remaining samples came from plants with vascular discoloration
and, of these, 10 tested positive for Foc TR4. This confirmed that
Results vascular discoloration in the trial was more than 99% accurate as
Generation and characterization of transgenic banana plants. a diagnostic marker for infection by TR4.
We have previously shown that the Ced9 anti-apoptosis gene By the end of the trial, between 67 and 100% of all control
derived from the nematode Caenorhabditis elegans can confer plants were either dead or infected and all had displayed vascular
resistance to Foc race 1 in transgenic Lady finger bananas8. We discoloration. In general, disease developed earlier in the non-
have also previously isolated resistance gene analog 2 (RGA2), a transgenic control plants, increased by about 20% per year, and
putative nucleotide-binding and leucine-rich repeat (NB-LRR)- was considerably faster during the wet seasons (Figs. 3a,b and
type resistance (R) gene, from a seedling of Musa acuminata ssp. Table 1). DPM25, which is noted for its resistance to Foc STR412,
malaccensis with resistance to TR49. Sequence analysis of this appeared to be the most susceptible cultivar with all of its
gene7 revealed a close phylogenetic relationship to the NB-LRR replicates becoming infected within 2.5 years, whereas the
type genes, including I210 and Fom-211, which have been shown reportedly TR4-tolerant GCTCV 2186 was as susceptible as the
to encode Fusarium wilt resistance in tomato and melon, Cavendish cultivar Williams. However, there was no statistical
respectively. Therefore, we constructed both Ced9- and RGA2- difference in the total proportion of symptomatic infected plants
derived transgene expression cassettes (Fig. 1a,b) where Ced9 was between any of the controls compared with the reference control,
a
LB 35S-T nptII 35S-P Nos-P RGA2 Nos-T RB
b
LB 35S-T nptII 35S-P Nos-T Ced9 Ubi-P RB
c d
Ced9-17
Ced9-19
Ced9-21
Ced9-22
Ced9-26
RGA2-2
RGA2-3
RGA2-4
RGA2-5
RGA2-7
WT
WT
23,130 bp
23,130 bp
9,416 bp
9,416 bp
6,557 bp
6,557 bp
4,361 bp 4,361 bp
RGA2 homologs
2,322 bp
2,027 bp
2,322 bp
2,027 bp
Fig. 1 Transgene expression cassettes and Southern analysis of selected transgenic lines. a RGA2 and b Ced9 expression cassettes. LB, left border; RB, right
border. Determination of transgene copy number in c RGA2 and d Ced9 transgenic banana lines by Southern blot analysis. Genomic DNA from WT, RGA2
and Ced9 lines was digested with HindIII and XmaI, respectively. DNA molecular weight marker II (Roche) reference is indicated on the right hand side
GN (Tukey's honest significant difference (HSD) test, p > 0.05). of RGA2 mRNA, whereas the other three TR4-resistant lines,
In contrast, 30% or fewer of plants of four of the five RGA2-2 (20% infection), RGA2-4 (20% infection), and RGA2-5
characterized RGA2 lines and four of the five characterized (14.3% infection), also showed moderate to high levels of RGA2
Ced9 lines were symptomatic during the 3-year trial. Symptom expression. The most susceptible line RGA2-7 (60% infection)
development in the four RGA2 lines, RGA2-2, RGA2-3, RGA2-4, had the lowest expression levels, which were about tenfold less
and RGA2-5, was significantly lower than that in GN (Tukey's than RGA2-3. The level of expression in the highly susceptible
HSD test, 0.01 < p < 0.05), whereas RGA2-7 (60%) was not. Cavendish cultivar GN (87.5% infection) was also about tenfold
Similarly, symptom development was significantly lower in the lower than line RGA2-3. The observed inverse correlation
four Ced9 lines, Ced9-22, and Ced9-26 (Tukey's HSD test, 0.01 < between the proportion of infections and the expression level of
p < 0.05), and Ced9-19 and Ced9-21 (Tukey's HSD test, 0.001 < RGA2 in non-transgenic GN and the RGA2 transgenic lines was
p < 0.01) than GN, whereas Ced9-17 (87.5%) was not. Lines statistically significant (Pearson's correlation = −0.86, t = −3.37,
RGA2-3 and Ced9-21 appeared to be immune to TR4, as no df = 4, p = 0.028; Spearman’s rank correlation = −0.90, S = 66.45,
internal (vascular discolouration) or external symptoms of the p = 0.015). This inverse relationship persisted when only the
disease were observed in any plant throughout the 3 years RGA2 transgenic lines were considered but the correlation was
(Figs. 2c–f, Figs. 3a, b, and Supplementary Table 1). no longer statistically significant (Pearson's correlation = −0.85,
t = −2.811, df = 3, p = 0.067; Spearman’s rank correlation =
−0.82, S = 36.42, p = 0.089).
Analysis of gene expression levels. To investigate the basis of the Interestingly, RGA2 expression in TR4-resistant wild diploid
resistance, and because TR4 is a soil-borne pathogen, roots from M. acuminata ssp. malaccensis was more than fivefold greater
the Ced9 and RGA2 lines were assayed for transgene expression. than in its susceptible sibling but more than fivefold less than
Quantitative analysis was only done on RGA2 lines, because, as a RGA2-3. Fortunately, enhancing levels of RGA2 or Ced9
banana-derived gene, RGA2was considered to be the most sui- expression does not appear to have a detrimental impact on
table transgene for deregulation. Reverse transcriptase PCR (RT- bunch size in the transgenic lines. A visual rating of the size of
PCR) showed that all five Ced9 lines expressed the transgene available mature bunches (Supplementary Table 2) based on the
(Supplementary Fig. 1). For the RGA2 lines, quantitative RT-PCR number of fruit hands per bunch showed no statistical difference
(qRT-PCR), initially with primers that amplified the RGA2 between healthy control and transgenic GN (χ2 = 31.7, df = 28,
transgene only, was used to assess expression levels and a strong p = 0.29).
correlation was observed between RGA2 expression level and the
degree of TR4 protection (Fig. 3c). Subsequently, qRT-PCR was
repeated but with primers that would amplify both the RGA2 Possible resistance mechanisms and future research. These
transgene and endogenous RGA2 in GN (Fig. 3d). Again, the results indicate that the Cavendish cultivar GN possesses endo-
most resistant transgenic line (RGA2-3) was the highest expressor genous RGA2 loci, which are not sufficiently expressed to confer
b d f
Fig. 2 Characteristic symptoms of Foc TR4 in susceptible and resistant banana. External symptoms and reddish-brown internal vascular discoloration of
Foc TR4 in infected WT Cavendish a and b compared with resistant transgenic lines RGA2-3 c and d, and Ced9-21 e and f
TR4 protection. Given the close relationship of GN and the other that are suitable for progression through to commercial release.
Cavendish cultivars Williams, GCTCV 218, and DPM25, it is Further, the discovery that Cavendish encodes three RGA2
probable that the latter clones also possess RGA2 loci. Increasing homologs provides the exciting prospect of using gene editing
RGA2 levels by transgenesis may reduce TR4 infection, possibly technology to generate TR4 resistance by enhancing the
through an R-gene-like cascade pathway. The gene(s) responsible expression of these endogenous genes with or without also
for the Foc TR4 resistance in M. acuminata ssp. malaccensis have editing the coding sequence.
not been identified but RGA2 is a possible candidate. It seems
highly unlikely that the observed resistance to TR4 in our RGA2 Methods
lines is due to somaclonal variation, because somaclonal variants Plant transformation and characterization. Binary vectors containing either the
that exhibit even tolerance to TR4 are rare, yet we identified four C. elegans gene Ced9 under the control of a maize Ubi-P and a cauliflower mosaic
out of five independent RGA2 lines with resistance. Southern blot 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
analyses indicated that even the highly susceptible GN contains were used in this study M. acuminata Cavendish cv. GN (AAA subgroup)
three RGA2-like sequences (Fig. 1c). Cloning and analysis of these embryogenic cell suspensions were prepared from immature male flowers and
RGA2-like sequences confirmed the presence of three RGA2 transformed using the centrifugation assisted A. tumefaciens-mediated method18.
homologs, which were between 98.6 and 98.7% homologous with Following selection, plants derived from single embryos were regenerated and were
screened for the presence of the respective transgene by PCR using specific primers.
RGA2 at the nucleotide level resulting in between 28 to 32 amino A total of six RGA2 and nine Ced9 lines were generated, and up to 10 replicates of
acid changes (Fig. 4). Further analysis indicated that these were each transgenic line was multiplied in tissue culture for field analysis. Both the
three alleles of a single copy RGA2 homolog. To determine number of transgenic lines and the number of replicates per line permitted in the
whether low expression levels of the homologs, amino acid dif- 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
ferences, or a combination of the two renders the three RGA2 suspensions were also generated as controls. Before field planting, tissue-culture
homologs ineffective, GN has been transformed with each plants were acclimatized in a secure shade house over a 3-month period by which
homolog, under the control of the Nos promoter, and the time they had reached a height of ~35 cm.
response of the transgenic lines to TR4 will be assessed in future
field trials. The mode of action of Ced9-mediated TR4 resistance Field trial design. The field trial was conducted on a commercial banana plan-
in transgenic bananas is also unclear. Functioning as an anti- tation site located at Lambells Lagoon, Northern Territory, Australia. The site had
apoptosis gene, it may be preventing fungal-induced cell death 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
and contributing to the maintenance of organelle homeostasis. January 2012 and the second in May 2012. Owing to the seriousness of the threat of
Previously, transgenic resistance to Foc race 1 in banana has TR4 to the Australian banana industry, it took 8 years from the initial generation of
been reported from glasshouse testing using either anti-apoptosis the transgenic lines to identify a suitable field trial location and obtain permission
genes8,15 or RNA interference targeting essential Foc genes16. from the plantation owner, the biosecurity regulators in both Queensland and the
Northern Territory, and the OGTR to conduct this trial. Unfortunately, the trial
This is the first report of Fusarium wilt resistance in transgenic was only conducted for 3 years, not the intended 5 years, because of a forced
bananas in the field. It is a very significant step toward averting quarantine termination order due to another disease.
the collapse of Cavendish-based banana export production and at Transgenic lines were planted in a randomized design, with rows containing
the same time protecting a major subsistence crop. We are about 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
to commence another more expansive field trial containing our selections GN and/or Williams) from QUT in addition to tissue culture plants of
RGA2 lines, as well as many more new Cavendish (cultivars Williams, GCTCV 218 and DPM25 supplied by Mission Beach Tissue Culture
Williams and GN) lines transformed with RGA2, to identify lines Nursery, Queensland. To increase the level and uniformity of inoculum pressure, a
a c
100 1.2
GCTCV 218
90
50 RGA2-3* 0.6
RGA2-4*
40
RGA2-5* 0.4
30
RGA2-7
20
0.2
10
0 0
Mal-S
Mal-R
GN
RGA2-2
RGA2-3
RGA2-4
RGA2-5
Jan-12 Jan-13 Jan-14 Jan-15
b d 1.2
100
Grand Nain
90
Ced9-21** 0.8
60 Ced9-22*
50 Ced9-26* 0.6
40
0.4
30
20
0.2
10
0 0
Mal-S
Mal-R
GN
RGA2-2
RGA2-3
RGA2-4
RGA2-5
RGA2-7
Jan-12 Jan-13 Jan-14 Jan-15
Wet season Wet season Wet season Wet season
Fig. 3 Disease incidence and gene expression analysis. a, b Levels of Foc TR4 infection in selected transgenic and WT banana plants throughout the 3-year
field trial. a WT and RGA2 lines, and b WT and Ced9 lines. Wet seasons (November–April) are indicated. The number of biological replicates (n) of each
independent line at the start of the trial is provided in Supplementary Table 1. Data points are percentage of biological replicates infected at time of
assessment. *0.01 < p < 0.05, **0.001 < p < 0.01 at trial end (Tukey's HSD test). c, d Analysis of RGA2 mRNA expression levels in transgenic and WT
banana plants. c Analysis of transgene (RGA2-Nos) expression levels using primers designed to amplify a 96 bp fragment spanning the RGA2 transgene/
Nos terminator junction. d Analysis of RGA2 transgene and endogenous mRNA expression levels using primers designed to amplify a 92 bp fragment of
both the RGA2 transgene and RGA2 endogenous sequences. All values are normalized expression levels expressed relative to line RGA2-3. WT GN; TR4-
susceptible M. acuminata ssp. malaccensis (Mal-S) and TR4-resistant M. acuminata ssp. malaccensis (Mal-R). A single biological replicate was analyzed with
three technical replicates (n = 3). Data are mean ± SEM
small segment of pseudostem taken from TR4-infected banana plants growing vascular discoloration was observed, a pseudostem sample of ~3 cm × 1 cm was
outside the trial site was buried between each plant. taken from the leading edge of the discoloration. Where no vascular discoloration
When a banana is planted, the plant crop, a pseudostem comprising the petioles was observed, a similar sample was taken from an equivalent area. Pseudostem
of leaves grows from the basal corm with the vegetative meristem remaining at the samples from 118 plants were collected and were sent by air freight to Wageningen
base of the pseudostem. When flowering is initiated, the meristem is pushed up UR, Plant Sciences Group, The Netherlands, for confirmatory diagnosis of Foc
through the center of the pseudostem, the fruit bunch emerges through the top of infection.
the pseudostem and develops through to maturity. After the bunch is harvested, To avoid any bias, all plants in the trial were assigned a unique Field Trial
this initial pseudostem dies back and another new pseudostem, known as the first Number which contained no identifying information. Further, the assessment of
ratoon, grows from a different meristem on the corm. This process can be repeated disease symptoms during the 3 year trial period was done independently by the
indefinitely. The trial consisted of the plant crop and up to three ratoon crops. 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).
Assessment of symptoms and bunch size. Plants were regularly assessed over At various times during the trial, the size of mature fruit bunches on healthy
three crop cycles (~3 years) for the development of characteristic external symp- transgenic and non-transgenic plants was visually assessed and rated into three
toms of Fusarium wilt disease1. The pseudostem of plants exhibiting typical categories, <6, 6–8, or >8 hands per bunch.
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 Diagnosis of Foc infection. The pseudostem samples were used for a combination
leaves, it almost invariably develops severe vascular necrosis followed by death of of fungal isolation and molecular diagnostic analyses by PCR13,14 and quantitative
that pseudostem. In some instances, the symptomatic pseudostem would die but an PCR (qPCR kit: “Foc TR4 DNA identification by Real-Time PCR, Clear Detec-
apparently healthy ratoon pseudostem would grow from the basal corm. The first tions”, The Netherlands). For DNA isolation from plant samples, small pieces of
observation of symptoms was recorded for each plant; however, ratoons from pseudostem tissue were selected and subsequently lyophilized in an Epsilon 1-4/
diseased plants were allowed to develop naturally until the completion of the trial 2–4 LSC plus freeze dryer (Martin Christ) for 3 days. For a subset of 49 samples
period. At the completion of the trial period, all plants were inspected for external that were collected from the above mentioned materials, 2–4 pieces of the same
and internal symptoms. The presence of the internal vascular discoloration, which tissue were sterilized with 1% hypochlorite, rinsed with Milli-Q water, dried in filter
is highly characteristic of Foc TR4 infection, was assessed in all remaining plants by paper, and placed on Komada medium for fungal re-isolation13. After 5 days,
cutting the pseudostem of all plants ~0.5 metres above ground level. Where emerging Foc colonies were sampled for PCR analysis13, as well as qPCR. Total
Table 1 Accumulated rates of Foc TR4 disease throughout the 3-year field trial
Line Number of plants in Chronological incidence of TR4 infection (percent of symptomatic plants)
the field (n)
January April October April October April October April
2012 2012 2012 2013 2013 2014 2014 2015
GCTCV 218 16 0 0 0 31.3 31.3 68.8 68.8 68.8
Williams 27 0 0 0 25.9 33.3 51.9 55.6 66.7
DPM25 9 0 0 22.2 77.8 88.9 88.9 100 100
GN 8 0 0 12.5 37.5 50 62.5 75 87.5
RGA2-2 10 0 0 0 0 0 0 0 20*
RGA2-3 8 0 0 0 0 0 0 0 0*
RGA2-4 10 0 0 0 0 0 0 0 20*
RGA2-5 7 0 14.3 14.3 14.3 14.3 14.3 14.3 14.3*
RGA2-6 6 0 0 16.7 50 50 50 50 66.7
RGA2-7 10 0 0 10 20 40 50 60 60
Ced9-10 8 0 0 12.5 25 37.5 37.5 37.5 37.5
Ced9-15 10 0 0 0 10 10 20 20 50
Ced9-17 8 0 0 25 25 25 37.5 50 87.5
Ced9-19 10 0 0 0 10 10 10 10 10**
Ced9-21 9 0 0 0 0 0 0 0 0**
Ced9-22 10 0 0 0 10 10 10 30 30*
Ced9-23 10 0 0 0 10 10 10 10 20
Ced9-26 10 0 0 20 20 20 20 20 20*
Ced9-31 10 0 0 10 10 10 20 20 20
DPM25, Dwarf Parfitt Mutant; GCTCV, Giant Cavendish Tissue Culture Variants; GN, Grand Nain; HSD, honest significant difference; TR4, tropical race 4. Data are percentage of biological replicates
infected at time of assessment. *0.01 < p < 0.05, **0.001 < p < 0.01 at trial end (Tukey's HSD test).
1 10 20 30 40 50 60 70 80 90 100
Mal-R RGA2
GN allele 1 RGA2
GN allele 2 RGA2
GN allele 3 RGA2
110 120 130 140 150 160 170 180 190 200 210
Mal-R RGA2
GN allele 1 RGA2
GN allele 2 RGA2
GN allele 3 RGA2
220 230 240 250 260 270 280 290 300 310 320
Mal-R RGA2
GN allele 1 RGA2
GN allele 2 RGA2
GN allele 3 RGA2
330 340 350 360 370 380 390 400 410 420 430
Mal-R RGA2
GN allele 1 RGA2
GN allele 2 RGA2
GN allele 3 RGA2
440 450 460 470 480 490 500 510 520 530 540
Mal-R RGA2
GN allele 1 RGA2
GN allele 2 RGA2
GN allele 3 RGA2
550 560 570 580 590 600 610 620 630 640
Mal-R RGA2 110
GN allele 1 RGA2
GN allele 2 RGA2
GN allele 3 RGA2
650 660 670 680 690 700 710 720 730 740 750
Mal-R RGA2
GN allele 1 RGA2
GN allele 2 RGA2
GN allele 3 RGA2
760 770 780 790 800 810 820 830 840 850 860
Mal-R RGA2
GN allele 1 RGA2
GN allele 2 RGA2
GN allele 3 RGA2
870 880 890 900 910 920 930 940 950 960 970
Mal-R RGA2
GN allele 1 RGA2
GN allele 2 RGA2
GN allele 3 RGA2
980 990 1,000 1,010 1,020 1,030 1,040 1,050 1,060 1,070 1,080
Mal-R RGA2
GN allele 1 RGA2
GN allele 2 RGA2
GN allele 3 RGA2
1,090 1,100 1,110 1,120 1,130 1,140 1,150 1,160 1,170 1,180
Mal-R RGA2
GN allele 1 RGA2
GN allele 2 RGA2
GN allele 3 RGA2
1,190 1,200 1,210 1,220 1,230 1,233
Mal-R RGA2
GN allele 1 RGA2
GN allele 2 RGA2
GN allele 3 RGA2
Fig. 4 Protein sequence alignment of the RGA2 transgene sequence from TR4-resistant M. acuminata ssp. malaccensis (Mal-R) and the three consensus
RGA2 homologous sequences from WT GN. Amino acid differences are highlighted
plant and fungal DNA was extracted using the Sbeadex maxi plant Kit (LGC µL of ROX reference dye II (Takara), 3 µL of DNA template, and Milli-Q water in a
Genomics). DNA extractions from all plant and fungal samples were independently 7500 Real-Time PCR system (Applied Biosystems). Thermal cycling conditions for
repeated at least twice. Analytical PCRs were also technically repeated twice. qPCR amplification were an initial enzyme activation at 95 °C for 3 min, followed by 35
was conducted in a total volume of 20 µL reaction mixture containing 10 µL of cycles each consisting of 95 °C for 10 s, 63 °C for 60 s, and 72 °C for 30 s. Finally, for
Clear Detections PCR mix, 2 µL of Foc TR4 primer set, 3 µL of PCR enhancer, 0.2 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 (Promega), and heat-shock transformed into competent Escherichia coli XL-1 Blue
Biosystems). 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
Transgene expression analysis. Owing to quarantine restrictions, we were unable Miniprep DNA purification system (Promega) and digested using NotI to
to transport samples from banana plants within Australia for analysis. Therefore, identify clones with inserts of the expected size. Plasmids were then sequenced
transcript analyzes were done using samples taken from the original mother plants using the BigDye Terminator mix v3.1 (Applied Biosystems) according to the
stored in tissue culture at QUT, Brisbane, Australia. Tissue culture plants of wild- manufacturer’s instructions, with sequence reads generated at the Queensland
type (WT) TR4-susceptible and resistant M. acuminata ssp. malaccensis were used University of Technology Molecular Genetics Research Facility. Specific primers
as controls19,20. were used to completely sequence the inserts in both directions. Raw sequence
Total RNA was extracted from 200 mg of root tissue using a protocol21 that was reads were compiled into full-length sequences using the VectorNTI Advance
modified by increasing the tissue:extraction buffer ratio to 8, including a V11 software (Life Technologies) program ContigExpress, whereas sequence
centrifugation step (18,000xg for 5 min) before the initial solvent extraction and the alignments were carried out using AlignX. Consensus sequences of the WT
omission of phenol from all extraction steps. RNA (3 μg) was DNase-treated using GN RGA2 homologs were compared with the M. acuminata spp. malaccensis
an RQ1 RNase-free DNase Kit (Promega) and DNA-free RNA samples (1.8 μg) RGA2 sequence, to determine the level of nucleotide and amino acid sequence
were reverse-transcribed to complementary DNA using an oligo(dT)18 primer and similarity.
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, Statistical analysis. Differences in total proportions of infected plants between the
respectively. To ensure complete removal of potential gDNA contamination in our treatment and control groups, and between lines, were assessed using single and
samples before RT-PCR and qPCR, total RNA, DNase-treated RNA, as well as mixed effects generalized linear models and corresponding analyses of variance,
cDNA was tested by PCR using the cyclophilin (CYP) housekeeping gene primers assuming a binomial response and with the respective addition or subtraction of
(Supplementary Table 3). 0.5 for 0 or total infections.
Reaction mixes for RT-PCR contained 1 × GoTaq Green master mix (Promega), Pairwise comparisons between treatments and lines, adjusted for multiple
0.25 µM of each primer (Supplementary Table 3), diluted cDNA (2 µL), and comparisons, were evaluated using Tukey's HSD test. Model fit was assessed using
nuclease-free water in a final volume of 20 µL. Thermal cycling conditions included a χ2-test of deviance and the Akaike information criterion (AIC).
a 2 min denaturation step at 94 °C followed by 35 cycles of 94 °C for 20 s, 55–62 °C The equivalence of ratings of fruit bunches from control, RGA2 lines and the
(primer dependent) for 30 s, and 72 °C for 1 min Kbp-1 of expected amplicon size, Ced9 lines as number of hands in the bunch (<6, 6–8, and >8) was assessed using a
and a final extension at 72 °C for 5 min. χ -test of homogeneity.
2
qRT-PCR was performed on a CFX384 Touch Real-Time PCR Detection System The hypothesis of a (linear) relationship between the proportion of infections
(Bio-Rad) using the SYBR Green I technology. Per 10 μL reactions, 2.5 μL of diluted and the expression level for each of the RGA lines and the GN control line was
cDNA was added to 1 × GoTaq qPCR Master Mix (Promega) premixed with primers assessed using parametric (Pearson) and nonparametric (Spearman) correlations,
(Supplementary Table 3) at a final concentration of 0.2 μM. The following and the associated tests.
amplification program was used: Hot-Start polymerase activation at 95 °C for 2 min, Statistical significance was asserted at the 5% level (p < 0.05). The analyses were
followed by 45 cycles of 10 s denaturation at 95 °C and 30 s annealing/extension at undertaken in R using base statistical functions and the packages lme4, lmer, and
60 °C. At the end of each run, a dissociation curve was produced from 65–95 °C, to multcomp.
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 Data availability. The authors declare that all data supporting the findings of this
coefficient R2>0.98 and efficiencies >99% (Supplementary Fig. 2). All samples were study are available within the article and/or its Supplementary Information file. All
analysed in triplicate and each run included triplicate non-template control reactions relevant data are also available from the authors upon request.
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.
Received: 26 July 2017 Accepted: 5 October 2017
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).
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