Constitutively Expressed RB Gene Confers a High Level but Unregulated Resistance to Potato Late Blight
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The RB gene, which was cloned from the wild potato species Solanum bulbocastanum, confers a high level of broad spectrum resistance to various strains of Phytophthora infestans, the causal agent of potato late blight. The level of RB-mediated resistance is correlated with the amount of RB transcripts in transgenic potato lines containing RB gene(s) driven by its native promoter. To assess whether the level of RB-mediated resistance can be further enhanced by overexpression of the RB gene, multiple transgenic potato lines containing RB gene(s) driven by the cauliflower mosaic virus (CaMV) 35S promoter were developed. Surprisingly, all 35S::RB transgenic lines with one or several copies of the RB gene showed a similar level of late blight resistance. In parallel, a statistically similar amount of RB transcript was observed among all resistant transgenic lines with different copy numbers of the RB gene. In addition, the levels of RB gene transcription in the 35S::RB transgenic potato lines were the same or lower than in transgenic lines containing the RB gene driven by its native promoter. Thus, developing transgenic potato lines using RB with the native promoter will be the best approach to deploy this gene for combating late blight.
KeywordsRB gene 35S promoter Transgenic potato Late blight resistance
El gen RB, que fue clonado de la especie silvestre de papa Solanum bulbocastanum, confiere un alto nivel y amplio espectro de resistencia a variantes de Phytophthora infestans, el agente causal del tizón tardío de la papa. El nivel de la resistencia mediada por el RB esta correlacionada con la cantidad de transcriptos de RB en líneas de papa transgénica que contiene gen(es) RB impulsados por el promotor nativo. Para evaluar si el nivel de resistencia mediada por RB puede aumentarse más mediante su sobreexpresión, se desarrollaron múltiples líneas transgénicas de papa con gen(es) RB conducidos por el virus mosaico de la coliflor (CaMV) 35S como promotor. Sorpresivamente, todas las líneas transgénicas 35S::RB con una o varias copias del gen RB mostraron un nivel similar de resistencia al tizón tardío. Paralelamente, se observó una cantidad similar estadísticamente del transcripto RB entre todas las líneas transgénicas resistentes con diferente número de copias del gen RB. Además, los niveles de transcripción del gen RB en las líneas transgénicas de papa 35S::RB fueron los mismos o más bajos que en las líneas transgénicas con el gen RB impulsado por su promotor nativo. De aquí que el desarrollo de líneas transgénicas de papa usando RB con su promotor nativo será la mejor estrategia para utilizar este gen en el combate al tizón tardío.
Potato late blight, caused by the oomycete Phytophthora infestans, is the most destructive potato disease in the world. Late blight causes losses of up to $14 billion annually in developing countries (Haverkort et al. 2009) due to the reduced yield, lower quality of the product, diminished storability, and increased cost associated with disease control (Nowicki et al. 2012). In developed countries, frequent fungicide applications have been the main approach to controlling this disease, which is both costly and a cause of significant environmental concerns (Haverkort et al. 2009). Development of potato cultivars with adequate resistance to P. infestans to avoid losses to late blight has been a major goal for the potato breeding community. Numerous late blight resistance (R) genes have been mapped and/or cloned from wild Solanum species (Nowicki et al. 2012; Rodewald and Trognita 2013; Witek et al. 2016; Chen et al. 2018), and some have been introgressed into cultivated potato. However, most of R genes provide resistance only to specific P. infestans strains, and such resistance is often quickly lost due to the evolving population dynamics of the late blight pathogen (Wastie 1991).
Solanum bulbocastanum is a wild diploid species with a high level resistance to late blight (Helgeson et al. 1998). The geographical distribution of S. bulbocastanum overlaps with the genetic diversity center of P. infestans (Grunwald and Flier 2005). Co-evolution of these organisms in the same environment has enabled S. bulbocastanum to develop durable resistance against P. infestans (Niederhauser and Millis 1953). Resistance screening of the S. bulbocastanum accessions stored at the United States Potato Genebank (http://www.ars-grin.gov/nr6/) revealed that all accessions had similar high levels of resistance to late blight (James Bradeen, personal communication). Four R genes have been cloned from S. bulbocastanum, including RB (also known as Rpi-blb1) (Song et al. 2003; van der Vossen et al. 2003), Rpi-blb2 (van der Vossen et al. 2005), Rpi-blb3 (Lokossou et al. 2009), and Rpi-bt1 (Oosumi et al. 2009). All four R genes belong to the NBS-LRR (nucleotide binding site-leucine rich repeat) class of resistance gene. RB, Rpi-blb2 and Rpi-blb3 encode proteins that recognize distinct P. infestans effectors that may be conserved in various P. infestans strains (Champouret et al. 2009; Halterman et al. 2010; Lokossou et al. 2009; Oh et al. 2009; Chen et al. 2012). Thus, potato plants containing these genes show broad-spectrum resistance against a variety of P. infestans strains.
The RB gene with its native promoter has been transferred into various potato cultivars (Bradeen et al. 2009; Halterman et al. 2008; Kuhl et al. 2007). It has been demonstrated that RB triggers an HR in both potato foliage and tuber challenged with P. infestans (Chen and Halterman 2011; Gao et al. 2013). However, individual transgenic potato lines often showed different levels of late blight resistance. Interestingly, the RB-mediated late blight resistance is positively correlated with the amount of RB transcripts in the transgenic lines (Bradeen et al. 2009; Kramer et al. 2009). Consequently, to assess the possibility of further enhancing the level of resistance by constitutively overexpressing the RB gene, an RB gene construct driven by the cauliflower mosaic virus (CaMV) 35S promoter was developed and used to develop multiple 35S::RB transgenic lines. We demonstrate that the 35S::RB transgenic potato lines did not provide improved late blight resistance compared to transgenic potato lines using RB with the native promoter.
Materials and Methods
The potato cultivar Katahdin was transformed with a modified pMD1 vector, which contains the full length coding sequence of the RB gene driven by the CaMV 35S promoter. Transgenic Katahdin potato lines were generated using an Agrobacterium-mediated transformation method described previously (Bhaskar et al. 2008). In vitro clones of transgenic lines were propagated and maintained in tissue culture under a 16 h 23 °C/8 h 21 °C light/dark regime on a modified MS medium (Phytotechnology Labs, Shawnee Mission, KS) containing 3% sucrose and 0.2% phytogel. Transgenic lines and control plants were grown at the Biotron greenhouses of the University of Wisconsin-Madison, where plants were grown at 15 to 25 °C with a light (irradiance 500 μmol m−1 s−1) and dark cycle of 16 h and 8 h, respectively.
Southern Blot Hybridizations
Southern blot hybridization was performed according to published protocols (Stupar et al. 2002). Genomic DNA was isolated from leaf tissue using the CTAB method and was quantified using a Nanodrop (ThermoFisher Scientific, Hampton, NH). Approximately 15 μg DNA from each line was singly digested with EcoRΙ or HindIII. The digested DNA fragments were separated on a 0.8% agarose gel and were blotted on an N+ membrane (GE Healthcare Limited, Amersham Place, NA). The blots were probed with a 530 bp PCR fragment amplified from pMD1 using forward primers 5’TTTGTCAAGACCGACCTGTC and reverse primer 5’CCAACGCTATGTCCTGAT. This DNA probe hybridizes to the NPTII gene which is in the left border of the pMD1 vector. The blots were probed overnight at 65 °C, washed, and autographed with standard protocols (Sambrook and Russell 2001).
Late Blight Resistance Evaluation
The 35S:RB Katahdin lines along with the control lines; including Katahdin, S. bulbocastanum accession PT29, and transgenic Katahdin lines 951, 925 with full length genomic RB DNA (Kramer et al. 2009), were evaluated for late blight resistance in the Biotron greenhouses using P. infestans isolate US930287 (US-8 genotype, A-2 mating type). The inoculation and resistance evaluation were performed as described previously (Colton et al. 2006). All lines were grown in triplicate and placed in a mist chamber within Biotron greenhouse eight hours prior to inoculation. A daytime temperature between 17 and 19 °C and a nighttime temperature at 13–15 °C as well 100% humidity were set in the mist chamber. All lines used in inoculation studies were grown in triplicate and were randomly placed in a mist chamber five hours prior to inoculation. The mist chamber held a relative humidity of >90% for 24-h, a 16 h light period, a daytime temperature of 17 to 19 °C and a nighttime temperature of 13 to 15 °C. Three biological replicate inoculations were performed using the following sporangial concentrations: 6.8 × 104, 6.6 × 104, 6.7 × 104 sporangia ml−1, respectively. Measurements of foliar late blight were interpreted and scored according to the Malcolmson scale (Cruickshank et al. 1982). The scale was based on percent of foliage symptomatic and scores were as follows: 9 = no visible symptoms; 8 = <10% symptoms; 7 = 11–25%; 6 = 26–40%; 5 = 41–60%; 4 = 61–70%; 3 = 71–80%; 2 = 81–90%; 1 = >90%; 0 = 100% symptoms. Disease scores were recorded 4, 7 and 10 days after inoculation. An average score for resistance was determined using the three replicate plants of each clone in each inoculation experiment. Statistical analysis was performed using a t-test (two-sample, unequal variance, two-tailed distribution).
Quantification of RB Gene Transcription
Quantification of the RB transcript was performed on six independent 35S:RB lines along with the original Katahdin, and transgenic lines 951 and 925. Leaf samples were collected 1, 2, and 3 weeks after the tissue culture plants were transplanted into greenhouses. Leaf samples were also collected 5 h after the plants were placed in the mist chamber before inoculation, and again 1, 2, 3, and 5 days post inoculation (dpi). Three independent leaves were collected from each plant for RNA isolation. RNA was isolated using the Qiagen Plant RNeasy kit (Qiagen, Germantown, Maryland) according to the manufacturers’ instructions. To eliminate DNA contamination, total RNA samples were treated with TURBO DNA-free (Ambion, Austin, TX). First strand cDNA was synthesized from 2 μg DNase-treated total RNA using SuperScript III reverse transcriptase with random hexamers (Invitrogen, Carlsbad, CA). DYNAMO SYBR Green master mix (Finnzymes, New England Biolabs, Ipswich, MA) and MJ Research Opticon 2 (Bio-Rad Laboratories, Hercules, CA) were used for quantitative real-time (qRT) PCR assay. All lines for inoculation experiment were grown in triplicate. Three independent leaves were collected from each plant and were mixed for RNA isolation. The following protocol was performed for all qRT-PCR assays: 15 min at 95 °C, 40 cycles of 20 s at 94 °C, 20 s at the corresponding annealing temperature, 30 s at 72 °C, followed by a plate read, and then, a melting curve of 50 to 95 °C with 0.2 °C steps, hold for 2 s, followed by a final extension step of 10 min at 72 °C. Primers for the RB gene were: 5’-CACGAGTGCCCTTTTCTGAC-3’ and 5’-ACAATTGAATTTTTAGACTT-3’. The primers are located within the leucine-rich repeat (LRR) domain of the second exon of the RB gene with an amplicon of 213 bp. S. tuberosum Actin-97, TC164213 was used as a reference gene. Primers for the actin gene were: 5’-GATGGCAGACGGAGAGGA-3’ and 5’-GAGGACAGGATGCTCCTC-3’. All statistical analyses were performed for the delta Ct values using t-tests with unequal variance against the baseline (35S:RB Katahdin line L1 or week 1 WK1) performed in R statistical analysis environment (Yuan et al. 2006).
Copy Numbers of the RB Gene in 35S::RB Transgenic Potato Lines
Late Blight Resistance Evaluation of the 35S::RB Transgenic Lines
Late blight resistance evaluation was performed on whole plants of the eleven characterized 35S::RB transgenic lines along with resistant and susceptible controls in the Biotron greenhouses, where relative humidity of more than 90% was maintained to facilitate P. infestans growth and infection. Three individual plants of each line were included in each inoculation experiment, and three biological replicates of inoculation experiments were performed using P. infestans strain US930287 (US-8 genotype).
Three transgenic lines were susceptible to late blight in all three inoculation experiments. The mean resistance ratings of these three lines (L14, L19 and L20) were 3.00 ± 0.44, 2.89 ± 0.45, 2.11 ± 0.11, respectively. Interestingly, L20 contained 5 copies of the RB gene, whereas both L14 and L19 contained approximately 10 copies of the RB gene (Fig. 1).
Transcription of the RB Gene(s) in Transgenic Potato Lines
Our previous work showed that RB-mediated late blight resistance is correlated with transcript abundance of the RB transgene (Kramer et al. 2009). To evaluate the amount of RB transcripts in the 35S::RB lines, we conducted qRT-PCR analysis in six representative transgenic lines (L1, L5, L7, L8, L19, L20) that covered different copy numbers of the RB gene. Leaf tissues were collected 1 week (WK1), 2 weeks (WK2), and 3 weeks (WK3) after planting, from plants that received mist treatment, and from plants at 1 day, 3 days and 5 days post inoculation with P. infestans. The expression of the potato actin gene was used to normalize the relative abundance of the RB transcript within each line.
Activation of R gene-based defense can trigger a significant and energetically costly transcriptomic changes in host plants. Thus, R genes are ideally either silent or expressed at a very low basal level during the absence of their cognate pathogens. Indeed, expression of some NBS-LRR genes is either induced (Mohr et al. 2010; Yoshimura et al. 1998), or is enhanced only upon pathogen infection (Cai et al. 1997; Halterman et al. 2003; Levy et al. 2004; Liu and Ekramoddoullah 2011; Radwan et al. 2005). Overexpression of some NBS-LRR genes has proved to be deleterious to the plants, causing dwarfing, sterility and other growth defects (Stokes et al. 2002), or results in cell death caused by the R gene-induced hypersensitive response (Bendahmane et al. 2002). Interestingly, overexpression of some other NBS-LRR genes only enhances the disease resistance and does not cause visible phenotypic abnormalities (Cao et al. 2007; Oldroyd and Staskawicz 1998; Xiao et al. 2001). We did not observe any unambiguous abnormal phenotypes from the 35S::RB transgenic lines under the growing conditions in greenhouses. Interestingly, three 35S::RB transgenic lines were susceptible to late blight although RB transcripts were detected in these lines. It is possible that these lines, which contain multiple copies of the RB gene, produce only incomplete RB transcripts. These transcripts, however, can still be amplified by PCR. Similarly, loss of Prf-mediated tomato bacterial resistance was reported in transgenic tomato lines containing high copy numbers of the Prf gene (Oldroyd and Staskawicz 1998).
It is interesting to note that the RB expression levels in the 35S::RB transgenic lines developed in this study were not greater than those of transgenic lines 951 and 925 containing the native promoter (Fig. 4). These results suggest that the native promoter of the RB gene plays a key role in high and optimal levels of RB gene expression. Thus, transgenic RB lines using the native promoter should be used in deploying this gene in potato cultivars. We noticed that the increase of RB transcripts detected in lines 951 and 925 after inoculation with P. infestans was not as dramatic as observed in previous experiments (Kramer et al. 2009). The relatively low concentration of sporangia used in this study for inoculating potato plants (6.6×104 to 6.8×104 sporangia/ml) compared to previous experiments (8.6×104–13×104 sporangia/ml) may account for this difference in levels of RB transcripts. We have consistently observed that the different levels of late blight resistance detected among the RB transgenic lines with the native promoter were especially visible when a high concentration of sporangia was applied during greenhouse inoculation tests. In addition, we used a different P. infestans strain (US930287) in the current study. P. infestans strains may have differential competence for inducing/suppressing RB gene expression.
We thank James Bradeen and Dennis Halterman for valuable comments on the manuscript. This research was supported partially by Hatch funds to J.J. L.W. was partially supported by National Natural Science Foundation of China (NO.31300127) and Research Program of science and technology at Universities of Inner Mongolia Autonomous Region (NJZY12002). The experiments comply with the current laws of United States of America and People’s Republic of China in which they were performed.
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