Botanical Studies (2009) 50: 413-424.
molecular biology
A tobacco rattle virus-induced gene silencing system for a soil-borne vascular pathogen Ralstonia solanacearum
Fang-I HO1,3, Yong-Yi CHEN2'3, Yu-Mei LIN2, Chiu-Ping CHENG2 *, and Jaw-Fen WANG1*
1AVRDC-The World Vegetable Center, P.O. Box 42, Shanhua, Tainan 741, Taiwan
2Institute of Plant Biology and Department of Life SScience, National Taiwan University, Taipei 106, Taiwan (Receiver February 13, 2009; Accepted April 14, 2009)
ABSTRACT. Bacterial wilt (BW), caused by Ralstonia solanacearum, is a devastating soil-borne vascular disease of solanaceous crops worldwide. However, information on the defense mechanisms of Solanaceae against this bacterium is limited. In this study, we optimized a virus-induced gene silencing (VIGS) system to broaden the application of VIGS for the study of plant response to soil-borne vascular pathogens such as R. solanacearum. Due to the soil-borne and rapid xylem-routed infection features of BW, factors involved in Tobacco rattle virus-based VIGS system, such as Agrobacterium tumefaciens strains, plant ages, inoculation methods and plant genotypes, were evaluated. An optimized cotyledon agroinfiltration method was developed using phytoene desaturase gene as the marker. All tomato cultivars, but a few pepper and no eggplant cultivars tested showed good competence in gene silencing using the optimized VIGS protocol. Because tomato responses at spatial and temporal levels are critical for the outcome of R. solanacearum infection, spatial and temporal assessments of VIGS efficiency were carried out. The results showed that silencing of TGA2.2 in 9-day-old tomato plants initiated 5 days post inoculation in stembases and later in roots and then in young leaves. Moreover, a chimeric approach was shown to be effective for multi-gene silencing than mixing multiple Agrobacterium strains carrying individual target genes. With the established protocol, we were able to show that silencing COI1, an essential key player in the jasmonic acid (JA) signaling pathway, led to increase of R. solanacearum proliferation in stembases and mid-stems of 'Hawaii 7996', a tomato cultivar with durable resistance to BW. Our study provides the first demonstration for a positive role of the JA signaling pathway in tomato resistance to BW, and notably, is inconsistent with the reports in Arabidopsis. The involvement and possible interplays of other known defense signaling pathways in the tomato BW-defense network to R. solanacearum are discussed.
Keywords: Bacterial wilt; Jasmonic acid defense signaling pathway; Ralstonia solanacearum; Virus-induced gene silencing.
Abbreviations: BW, bacterial wilt; VIGS, virus-induced gene silencing; JA, jasmonic acid; CFU, colony-forming unit; QTL, quantitative trait loci; ET, ethylene; ABA, abscisic acid; siRNAs, small interfering RNAs; TRV, tobacco rattle virus; JAR1, jasmonate resistant 1; JAZ, jasmonate ZIM-domain; RT-PCR, reverse transcriptase-PCR; PDS, phytoene desaturase; sqRT-PCR, semi-quantitative RT-PCR; DPI, days post inoculation; MAPK, mitogen-activated protein kinase.
introduction
Bacterial wilt (BW), caused by Ralstonia solanacearum, is a severe soil-borne vascular disease commonly occurring in the warm, humid tropics. This pathogenic bacterium has a wide host range; solanaceous vegetables, including tomatoes, eggplants, and peppers,
3These authors contributed equally to this work.
^Corresponding authors: E-mail: jaw-fen.wang@worldveg. org, Tel: +886-6-583-7804 ext. 450, Fax: +886-6-583-0009 (Jaw-Fen WANG); E-mail: chiupingcheng@ntu.edu.tw,
Tel: +886-2-3366-2521; Fax: +886-2-2391-8940 (Chiu-Ping CHENG).
are among the crops that suffer the most severe yield loss (Denny, 2006). Host resistance is the best control strategy due to the pathogen's soil-borne nature and wide host range. However, breeding for durable resistance in tomato plants has been hindered by the presence of diverse pathogen strains, and only a few resistant sources and commercial cultivars are available (Scott et al., 2005). Thus, gaining insight into the natural defense mechanism against R. solanacearum could benefit and facilitate the development of materials with durable resistance. In the natural infection process, R. solanacearum invades tomato plants at the emergence sites of secondary roots or at root tips, propagates intercellularly, and then enters the xylem (Vasse et al., 1995). The infection then progresses
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s ystemically, accompanied by bacterial propagation and the secretion of a large amount of extracellular polysaccharide, leading to complete wilting (Denny, 2006). The known resistant genotypes in tomatoes, eggplants, and peppers are not immune to the pathogen (Denny, 2006). Latent infections occur in resistant cultivars (Prior et al., 1994). The visual wilting symptom caused by R. solanacearum in tomato plants is correlated with the pathogen distribution and density in the stem. In resistant genotypes, the pathogen is suppressed and cannot multiply to the high-enough population level (109-101() CFU g-1) required in plant tissues to cause the wilting symptom
(Grimault et al., 1994; Wang and Lin, 2005). It has been
shown that the pathogen is largely confined to the primary xylem and is a poor colonizer in the resistant tomato
variety Hawaii 7996 (H7996) (Nakaho et al., 2004). The
resistance in H7996 involved several quantitative trait loci (QTLs) and appeared to be complex (Wang et al., 2000;
Cameille et al., 2006).
Very little is known about plant defense mechanism in solanaceous plants against R. solanacearum. Current knowledge about defense mechanisms against R. solanacearum is mostly derived from the studies carried out in Arabidopsis. It has been demonstrated that jasmonic acid (JA), ethylene (ET) and abscisic acid (ABA) related defense signaling pathways are involved in Arabidopsis response to the bacterium's attack (Deslandes et al.,
2002; Hernandez-Blanco et al., 2007; Hirsch et al., 2002).
However, the information gained from the Arabidopsis system cannot be applied directly to the tomato system, as several studies have revealed that the molecular mechanisms and responses of solanaceous plants to R. solanacearum or other pathogens could be different from those in Arabidopsis (Robatzek et al., 2007; Wei et al., 2007; Lin et al., 2008). In this study, we established an effective gene silencing tool which would facilitate rapid gene functional assays to reveal the complex resistance mechanism in solanaceous hosts against R. solanacearum.
Virus -induced gene silencing (VIGS) (Burch-Smith et al., 2004) has been widely used as a powerful reverse-genetics tool in functional genomic studies. The advantages of VIGS include the transient nature and high efficiency in rapid knockdown of specific genes or gene families with functional redundancy (Burch-Smith et al., 2004). After plants are infected with the genetically modified viruses that carry a short sequence of a target gene in their genomes, specific small interfering RNAs (siRNAs) are generated resulting from enzyme-mediated degradation of the virus transcripts (Ossowski
et al., 2008). The siRNAs would then bind to other RNA
molecules containing their complementary regions to trigger further rounds of cleavage of target transcripts and consequently result in effectively silence the corresponding endogenous genes in host plants. VIGS has been successfully employed to study roles of genes in various aspects of plant function in a variety of species, including disease defense (Ekengren et al., 2003), abiotic response
(Yoshioka et al., 2003; Senthil-Kumar et al., 2007a), and growth and development (Ahn et al., 2006). Among VIGS systems established to date, Tobacco rattle virus (TRV)-based vectors are the most commonly used vectors for its special properties, including: (1) TRV-induced symptoms in plants are usually very mild and thus do not disturb the phenotypic alterations caused by silencing of the target genes; (2) Unlike most plant viruses, TRV is capable of invading most plant tissues and cells, including meristems (Liu et al., 2002); (3) The efficacy of TRV-VIGS systems seems to be beyond its reputed host range of our knowledge (reviewed in Ekengre et al., 2003). TRV-VIGS systems have been used for functional genetic studies carried out in a range of species, particularly in Solanaceae
(Ekengre et al., 2003; Senthil-Kumar et al., 2007).
TRV-VIGS has been used to study defense mechanisms of foliar diseases such as tomato bacterial speck (Ekengren et al., 2003), and root diseases such as root knot nematode
(Bhattarai et al., 2007). However, TRV-VIGS systems have
not been used in studies of complex interactions involving plant host and soil-borne vascular pathogens such as R. solanacearum. Because of its nature of soil-borne and rapid xylem-routed infection, the use of an efficient and rapid system to dissect the complex mechanisms involved in plant responses to the infection of this destructive bacterium would be certainly desired. Therefore, there is a need to evaluate and optimize important parameters of the TRV-VIGS system, including Agrobacterium strains, inoculation methods, and host plant genotypes. Additionally, as R. solanacearum invades the host from roots and cause systemic infection, and because plant responses at spatial and temporal levels are critical for the outcome of R. solanacearum infection (Wang et al., 2000), it is thus important to confirm the temporal and spatial silencing efficacy when carrying out VIGS assays. Moreover, silencing multiple genes to discriminate the complex parallel signaling pathway is desired, as the interactions between R. solanacearum and solanaceous hosts are expected to be complex (Chen et al., 2009). Chen et al. (2004) demonstrated the effectiveness of co-silencing of two genes using TRV containing a tandem construct to study functions of floral-associated genes in petunia. Similar protocols would be valuable for the study roles of resistance-associated genes in BW-resistant genotypes in response to R. solanacaerum infection.
To use the optimized TRV-VIGS protocol developed in this study for determining roles of candidate genes in BW-defense mechanisms of solanaceous crops, a functional assay of COI1 , a SCF-type E3 -ubiquitin ligase, was conducted in a tomato cultivar resistant to R. solanacearum. Being the receptor of JA signaling, COI1 plays an essential function in the JA signaling pathway and controls most JA responses (Xie et al., 1998; Li et al., 2004). In response to the developmental cues or environmental stimuli, plants produce bioactive amino acid-conjugated JAs from free form of JA by Jasmonate Resistant 1 (JAR1) enzyme. Binding of conjugated JAs
HO et al. 一 Optimizing TRV-VIGS system for systemic diseases
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to COI1 triggers further direct interaction of COI1 with Jasmonate ZIM-domain (JAZ) proteins. The subsequent ubiquitination and degradation of JAZs release the JAZ-mediated repression of early JA-responsive genes and JA response pathways is then switched on (Thines et al., 2007). The JA signaling pathway plays important roles in plant disease response; however its role varies among plant-pathogen systems (Kunkel and Brooks, 2002; Robert-Seilaniantz et al., 2007), and its role in tomato defense mechanism to R. solanacearum is not clear.
T h e a i m o f t h i s s t u d y w a s t o o p t i m i z e t h e T RV-VIGS conditions for rapid and reliable gene functional analyses for the study of complex interactions between solanaceous plants and R. solanacearum. Factors which may affect TRV-VIGS efficiency were analyzed, including A. tumeficiens strains, plant ages, inoculation methods, and solanaceous plant species and cultivars. Additionally, spatial and temporal efficacy of this system in tomato plants was determined and key factors affecting multiple gene silencing were examined. Furthermore, this optimized VIGS protocol was used to study the role of COI1 in tomato BW-defense mechanism. The optimization of the high-efficient TRV-VIGS system in tomato plants allowed a quick and the first demonstration for a positive role of the JA signaling pathway in tomato resistance to R. solanacearum.
materials and methods
Plant materials and growth conditions
S olanaceous plants used in this study included reported resistant (R) and susceptible (S) cultivars to R. solanacearum. Pepper (Capsicum annuum) cultivars
included: PBC3 84(R), PBC 53 5(R), PBC 066(R),
PBC631A(R), PBC743(R), Early Calwonder (ECW; S), and PBC1367(S). Eggplant (Solanum melongena)
cultivars included: EG219(R), EG192(R), EG203(R), S56B(R), S3(R), and EG048(S). Tomato (Solanum lyco-
persicum) cultivars included: H7996 (R), Hawaii 7997(R),
CRA66(R), BF Okitsu 101(R), R3034(R), L390(S),
and L305(S). Seeds were directly sown or after 2 day-priming in two-inch pots containing potting mixture (MOS-010; Known-You Seed Co.). Plants were grown in a growth chamber at 24°C with relative humidity from 60% to 80% and 16 hours extended daylight. 1000X liquid fertilizer (nitrogen: phosphorus: potassium 20:19:19; COMPO GmbH & CO. KG) was applied once before Agrobacterium infiltration. Plants were inoculated with Agrobacterium suspensions on the fully expanded cotyledons (9-10 days after sowing) or on the first true leaf (14 days after sowing).
Plasmid constructs and Agrobacterium tumefacisns strains
pTRV1 and pTRV2 derivatives were used as the vectors for gene silencing (Liu et al., 2002). Fragments of PDS, ACO5, TGA2.2 and COI1 were obtained by reverse
transcriptase-PCR (RT-PCR) from the tomato (H7996),
eggplant (EG203) or pepper (ECW) complementary DNA were used for experiments carried out in the respective plant species. Fragments of additional tomato genes tested in this work were amplified from tomato H7996 complementary DNA using primers listed in Table 1. The PCR product was cloned into the pCR® 8/GW/TOPO® vector. To prepare a chimeric construct of
PDS, TGA2.2, and ACO5, fragments of PDS and TGA2.2
were first digested with XbaI and/or SpeI, respectively, and ligated into pCR®8/GW/TOPO® vector to generate pCR®8/TGA2.2/PDS construct. Then XbaI and SpeI digested ACO5 fragment was cloned into pCR®8/TGA2.2/ PDS to generate a chimeric construct of pCR®8/TGA2.2/ PDS/ACO5. Finally, pCR®8/TGA2.2/PDS/ACO5 was then recombined into a pTRV2 vector, pYL279, by carrying out an LR recombination reaction using the Gateway®
system (Invitrogen Co., Carlsbad, CA, USA). pYL279-m,
a modified pTRV2 empty vector, in which the selectable
CHLORAMPHENICOL RESISTANCE gene and the
counter-selectable ccdB gene had been removed, was constructed via an LR recombination reaction with the empty pCR®8/GW/TOPO® vector. This was used as the empty vector control. The pYL279 recombinant constructs obtained were electroporated into cells of A. tumefaciens by a MicroPulser (Bio-Rad, USA). Three commonly used laboratory strains of A. tumefaciens, GV3101, GV2260 and C58C1, were used for transformation of the cloned silencing constructs in the indicated VIGS experiments.
Virus infection by Agrobacterium-mediated infiltration and agrodrench
A 3-mL culture of A. tumefaciens containing each TRV derivative was grown at 28°C in the appropriate antibiotic selection YEP medium (0.5% peptone, 0.5% yeast extract, and 1% sodium chlorine) containing 50 [ig/mL kanamycin, 50 ig/mL gentamycin, and 25 ig/mL rifamycin for overnight. The culture was then inoculated into a 25-mL YEP medium containing the antibiotics, 10 mM MES and 20 mM acetosyringone and grown overnight at 28°C with shaking. Agrobacterium cells were harvested and resuspended in an infiltration buffer (10 mM MgCl2, 10 mM MES, 200 mM acetosyringone, pH 5.6), adjusted to OD600 of 1.5-2.0. Suspensions of pTRV1 and pTRV2 were mixed in 1:1 ratio and incubated at room temperature for 3 h before inoculation. Agrobacterium suspension was infiltrated into two cotyledons or first true leaves using a 1-mL needleless syringe. For the agrodrench method, 20 mL of Agrobacterium suspension was drenched over the soil surface of each pot. Agrobacterium-infected plants were maintained at 22°C or 24°C in a growth chamber for the indicated periods of time. In experiments comparing the effects of Agrobacterium strains, inoculation methods, and plant cultivars, the experiment design used was randomized complete block design with 3 replications and 10 plants per replication. In the experiments involving PDS silencing, percentages of plants displaying the photo-bleaching phenotype were recorded periodically.
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When conducting analysis of variance, percentage data were transformed with arcsine square root and the least significant difference at 5% was used for mean comparisons.
Assessment of plant response to the infection of R. solanacearum
Ralstonia solanacearum strain Pss4 (phylotype I, race 1, biovar 3) was prepared to give an OD600 of 0.6 and used to challenge plants. The Pss4 suspension was poured over the pot soil surface of 3-week-old seedlings as described previously (Wang et al., 2000). The response of the inoculated plants were evaluated as the percentage of wilted plants 5 days after inoculation. When bacterial population in tomato plants were evaluated, stem were sterilized with 70% ethanol, and segments (about 1 cm in length) were removed from the base and mid-point of each plant, weighted and crushed in 1 mL of sterilized water, respectively. 100 of the tissue extract was subjected to 10-fold serial dilutions, and 10 [iL of each diluted sample was spotted onto modified SM1 medium. The number of bacterial colonies developed was recorded two days after incubation at 28°C as described previously (Lin et al., 2008). The detection limit of this method is about 103 CFU/g tissues. Each assay included about 6 to 12 plants and repeated at least three times. Pair-wise mean comparisons of bacterial population at the stembases and mid-stems were conducted between TRV-infected and silenced plants with Student t-test.
Semi-quantitative RT-PCR analysis
Total RNA was isolated from roots, collars or stems with the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany). One [ig of total RNA was treated with RNase-free DNase I (Qiagen) followed by phenol/chloroform extraction to remove DNase. The first-strand cDNA was synthesized from 0.1 mg of total RNA with oligo (dT) primer and AMV reverse transcriptase (Promega, Madison, WI, USA). Semi-quantitative RT-PCR was performed as described (Ekengren et al., 2003). Primers used for RT-PCR to check transcript abundance of the silenced genes are listed in Table 1. Tomato ubiquitin gene Ubi3 served as an internal control for equal cDNA use from control and silenced plants. To confirm lack of genomic DNA contamination, 200 ng of DNase I-treated RNA was also used as template for PCR reactions. The amplified products were analyzed on 1% (w/v) agarose gels stained with ethidium bromide.
results
Effects of inoculation methods, Agrobacterium strains and solanaceous species and cultivars on VIGS efficiency
VIGS efficacy of agroinfiltration on cotyledons and first true leaves was evaluated using Agrobacterium tumefaciens strains GV3101 or C58C1 as the carrier
  
HO et al. 一 Optimizing TRV-VIGS system for systemic diseases
417
bacterium and tomato phytoene desaturase (PDS) gene (Table 1) as a marker gene. The results showed that both methods and strains could efficiently cause the typical leaf bleaching phenotype on all silenced tomato plants and the silencing progression was similar among treatments (Figure 1A). Similar results were observed on two tomato cultivars, H7996 and Hawaii 7997. In addition, an agrodrench method, using A. tumefaciens strain GV2260 as reported previously (Ryu et al., 2004), resulted in leaf bleaching on 10% of the silenced plants at the end of the experiments (17 days after agrodrench), while agroinfiltration on cotyledons with A. tumefaciens
strain GV3101 or C58C1 could lead to leaf bleaching
on all of the silenced plants 14 days after agroinfiltration (Figure 1 B). Considering the desired plant age for following inoculation with R. solanacearum, the cotyledon agroinfiltration method with strain GV3101 was used for further studies.
Silencing efficiency of the cotyledon agroinfiltration method was evaluated on a set of resistant and susceptible cultivars of tomatoes, peppers and eggplants using PDS as a marker gene (Table 2). PDS inserts originated from tomato H7996, pepper ECW, and eggplant EG203 were used in respective VIGS assays. The results showed that the seven tomato cultivars displayed similar competence
for efficient VIGS, except L305 and R3034 had lower
percentage of bleached plants. Among the tested pepper cultivars, the best silencing result was observed on
PBC1367, followed by ECW and PBC535, while the other
four cultivars were not competent for efficient VIGS. All of the six eggplant cultivars tested displayed poor or no silencing effect using the protocol.
Spatial and temporal analysis of VIGS efficiency
Because tomato responses at spatial and temporal levels are critical for the outcome of R. solanacearum infection,
t o o o o o
A 1
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Figure 1. Effects of A. tumefaciens strains, plant ages, and inoculation methods on VIGS efficiency. Cotyledon infiltration was conducted on 10-day-old tomato H7996 plants (#) and first true leaf infiltration on 14-day-old plants ) with A.
tumefaciens strain GV3101 or C58C1 containing pTRV1 plus pTRV2-PDS (A); Cotyledon infiltration method was compared with agrodrench inoculation on 10-day-old plants with GV2260 strain (B). Silencing efficiency was evaluated based on the percentage of plants showing photo-bleaching phenotype in newborn leaves. The experiment was performed three times with 10 plants per treatment. Data presented were means over experiments.
Table 2. PDS silencing efficiency on tomato, pepper or eggplant cultivars resistant or susceptible to R. solanacearum.
Tomato
Pepper
Eggplant
Cultivara
Bleached plant (%)b
Cultivara
Bleached plant (%)b
Cultivara
Bleached plant (%)b
L390 (S)
100.0 (89.7)
PBC1367 (S)
96.7 (83.6)
S3 (R)
6.7 (9.1)
BF Okitsu 101 (R)
100.0 (89.7)
ECW (S)
76.7 (66.0)
S56B (R)
3.3 (6.3)
H7996 (R)
96.7 (83.6)
PBC535 (R)
53.3 (46.9)
EG219 (R)
0.0 (0.3)
CRA66 (R)
96.7 (83.6)
PBC384 (R)
10.0 (15.1)
EG192 (R)
0.0 (0.3)
H7997 (R)
93.3 (77.6)
PBC066 (R)
6.7 (12.4)
EG203 (R)
0.0 (0.3)
L305 (S)
76.7 (64.1)
PBC631A (R)
0.0 (0.3)
EG048 (S)
0.0 (0.3)
R3034 (R)
73.3 (61.2)
PBC743 (R)
0.0 (0.3)
LSD 0.05
(22.4)
LSD 0.05
(19.4)
LSD 0.05
(12.3)
a Reported resistant (R) or susceptible (S) cultivars. Cotyledons of 10-day-old plants were agroinfiltrated with A. tumefaciens strain GV3101 containing pTRV1 plus pTRV2-PDS. PDS inserts originated from tomato H7996, pepper ECW and eggplant EG203 were used in respective VIGS assays.
b Data of the percentage of bleached plants recorded 23, 26, 30 days after agroinfiltration for the tomato, pepper and eggplant cultivars, respectively, were transformed by arcsin square root for ANOVA. Transformed data were presented in the parentheses. LSD0 05 was calculated using the transformed data.
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Botanical Studies, Vol. 50, 2009
whether and when the cotyledon agroinfiltration method could effectively trigger systemic gene silencing effect upward and downward at the transcriptional level was further examined. Results of semi-quantitative RT-PCR (sqRT-PCR) illustrated that silencing of tomato PDS in roots and young leaves was efficiently achieved using our TRV-VIGS protocol (data not shown). Because we are interested in studying plant defense response to pathogen infection, tomato TGA2.2 (Table 1) was used as a marker gene for further analysis. TGA2.2 is a key transcription factor downstream of NPR1, the central regulator of salicylic acid and JA defense signaling pathway (Ekengren et al., 2003). The results revealed that the reduction of TGA2.2 transcript level in 10-day-old plants initiated 5 days post inoculation (DPI) in stembases (Figure 2A), 7 DPI in roots (Figure 2B) and 10 DPI in young leaves (Figure 2C).
VI GS-m edi ated si mu ltan eou s si l en ci n g of multiple genes
Factors involved in TRV-based VIGS were tested further to determine how VIGS-based multiple-gene silencing can be efficiently achieved. In general, two approaches can be used to silence multiple genes simultaneously by VIGS. O ne approach would be inoculation of plants with a mixture of several Agrobacterium lines, in which each line carries individual target genes. Therefore, the effect of Agrobacterium titer used for agroinfiltration was first assessed. As shown in Figure 3, when A. tumefaciens strain GV3101 containing pTRV1 plus pTRV2-PDS was diluted with the inoculation buffer, the VIGS efficacy of diluted Agrobacterium samples, even at an 8-fold dilution, remained the same as that of the original dose. However, when A. tumefaciens strain GV3101 containing pTRV1 plus pTRV2-PDS was diluted with Agrobacterium carrying empty TRV vector, the VIGS efficacy significantly reduced.
Another approach to achieve multiple-gene VIGS would be agroinfiltration of plants with a single Agrobacterium carrying a pTRV2 vector containing chimeric sequences from the targeted genes. We hence compared the efficiency of the mixing approach and the chimeric approach for multiple-gene VIGS. In this analysis, three genes with distinct functions were included (Table 1): PDS (a commonly used marker gene for VIGS and functioning in pigmentation), ACO5 (involved in ethylene biosynthesis), and TGA2.2 (playing an important role in disease defense). The results showed that PDS alone and the chimeric construct caused efficient leaf bleaching on all of the silenced tomato plants 17 DPI, while only 23% of the silenced plants showed bleaching phenotype when a mixing approach was used (Figure 4A). When the VIGS efficacy was analyzed spatially at the transcriptional level, sqRT-PCR results illustrated the reduced level of PDS transcripts caused by VIGS with a chimeric construct was not apparently different from PDS alone in stembases, roots and young leaves (Figure 4B); however, VIGS by mixing multiple Agrobacterium strains
failed to reduce the PDS transcript level in any of the tissues. Together, these results indicate that the chimeric approach is more efficient for multiple-gene silencing compared to the mixing approach.
Silencing of COI1 resulted in increase of R. solanacearum proliferation in tomato H7996 plants
The cotyledon agroinfiltration protocol developed above was used to evaluate whether COI1 is involved
Figure 2. Spatial and temporal analysis of VIGS efficiency. Cotyledons of 9-day-old tomato H7996 plants were agroinfiltrated with A. tumefaciens strain GV3101 containing pTRV1, pTRV2-TGA2.2, or pTRV2 empty vector. pTRV1 plus pTRV2-TGA2.2 is referred to as silenced plants and pTRV1 plus pTRV2 empty vector is referred to as TRV-infected plants. cDNA was synthesized from total RNA isolated from pooled samples of stembases (A), roots (B) or young leaves (C) collected from two plants agroinfiltrated with TRV or TRV-TGA2.2 after 5, 7 or 10 days. Primers used for TGA2.2-specific amplification are listed in Table 1. The experiment was performed twice with similar results. PCR amplification from cDNA from a single representative sample is presented. Amplification of tomato ubiquitin gene Ubi3 was used as an internal control for equal cDNA use from control and silenced plants. PCR cycles are indicated on the top of the sections. Lane M indicates DNA ladder and NC indicates negative control where RNA was used as template for PCR amplification in the absence of reverse transcriptase.
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when the internal R. solanacearum density reached a mean greater than 109 CFU g-1 in midstems. The COI1-silenced H7996 plants did not develop apparent wilting symptoms 12 days after R. solanacearum inoculation. However, when compared to the control plants, a much higher proportion of COI-1-silenced plants were detected with the presence of the bacterium in stembases (60.0% vs. 27.3%) and midstems (23.3% vs. 0.0%). Large variations in the bacterial density of assayed H7996 plants were observed. This is due to several reasons, including the high detection limit of the direct plating method (> 103 CFU g-1 plant tissues), the effect of plant physiological status and environment on the progress of the disease, and the uneven gene silencing degree over tested plants and even within
 
Figure 3. Effects of Agrobacterium inoculum titer on VIGS efficiency. Cotyledons of 10-day-old H7996 plants were agroinfiltrated with A. tumefaciens strain GV3101 containing pTRV1 plus pTRV2-PDS diluted at various folds with the inoculation buffer or Agrobacterium carrying the pTRV2 empty vector. T0: original concentration of pTRV2-PDS. T1, T2 and T3: pTRV2-PDS was diluted at 2-, 4國and 8-fold with the inoculation buffer. T4, T5 and T6: pTRV2-PDS was diluted at 2-, 4- and 8-fold with Agrobacterium carrying the pTRV2 empty vector. The silencing efficiency was evaluated based on the percentage of plants showing photo-bleaching phenotype in newborn leaves. The experiment was performed three times with 10 plants per treatment. The percentage data were transformed by arcsine square root for mean comparisons. Letters indicate significant differences on mean comparison by using Fisher's LSD test at p =0.05.
in the disease-defense mechanism of tomato H7996. As illustrated in Figure 5A, the accumulation of COI1 transcripts in stembases of COI1-silenced plants had been reduced 15 days post Agrobacterium infiltration. No noteworthy sequence homology (21 continuous identical nucleotides) was found between the COI1 fragment and ESTs or genes currently available in tomato sequence databases (Thomas et al., 2001; Ekengren et al., 2003; Bhattarai et al., 2007; Senthil-Kumar et al., 2007a). No apparent alteration in plant morphology was observed.
Resistance to R. solanacearum in tomato plants is usually correlated with the pathogen distribution and density in the stem (Grimault et al., 1994, Wang and Lin 2005). Therefore, both visual symptom development and internal bacterial density in stems were determined in this experiment. COI1-silenced H7996 plants, along with plants agroinfiltrated with the empty TRV vector, were inoculated with R. solanacearum by root drenching 10 days post agroinfiltration, when VIGS efficacy already has started in roots, the stembases, and even in young leaves. Tomato cultivar L390 was used as the susceptible control to confirm the success of pathogen inoculation. Under the defined experimental condition, we observed that TRV infection slightly slowed plant growth of H7996 and L390, but their response to the infection of R. solanacearum was not different from the control plants without VIGS treatment (data not shown). L390 plants first showed wilting symptoms 3 DPI and completely wilted 5 DPI,
Figure 4. VIGS-mediated simultaneous silencing of multiple genes. H7996 plants were agroinfiltrated with A. tumefaciens strain GV3101 containing pTRV1 plus various pTRV2 vectors.
PDS, pTRV1 plus pTRV2-PDS; chimeric, pTRV1 plus chimeric
pTRV2-PDS-TGA2.2-ACO5; mixed, pTRV1 plus a mixture containing equal amount of pTRV2-PDS, TRV2-TGA2.2 and pTRV2-ACO5. (A) Leaf photobleaching caused by PDS gene silencing. The VIGS efficiency was evaluated based on the percentage of plants showing photo-bleaching phenotype in newborn leaves. The experiment was performed three times with 10 plants for each treatment in each independent experiment. The percentage data were transformed by arcsine square root for mean comparisons. Letters indicate significant differences on mean comparison by using Fisher's LSD test at p =0.05; (B) VIGS-mediated reduction of PDS transcript levels. cDNA was synthesized from total RNA isolated from pooled samples of roots, stembases, or young leaves collected from two plants 15 days after agroinfiltration. Primers used for PDS-specific amplification are listed in Table 1. Amplification of tomato ubiquitin gene Ubi3 was used as an internal control for equal cDNA use from control and silenced plants. PCR cycles are indicated on the top of the sections. Lane M indicates DNA ladder and NC indicates negative control where RNA was used as template for PCR amplification in the absence of reverse transcriptase.
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the same plant. Nevertheless, pair-wise t-test showed that the COI-1-silenced plants had significantly higher bacterial population in stembases (4.38 vs. 1.89 log CFU g-1 tissue) and mid-stems (1.90 vs. 0.00 log CFU g-1 tissue) compared to that of H7996 plants inoculated with the empty vector (Figure 5B). These results indicate that silencing the JA signaling pathway increased the population of R. solanacearum in the resistant tomato plants.
discussion
TRV-based VIGS systems have been widely used in functional genomics studies for plants; however, it has not
been used for studies of plant defense mechanisms to soil-borne vascular pathogens, such as R. solanacearum. To establish an optimized method for such studies, we first evaluated factors that may affect TRV-VIGS efficiency, and confirmed the system's spatial and temporal efficacy in tomato plants.
Plant age is a main concern when establishing the TRV-VIGS protocol, as it is known that plants become more resistant to R. solanacearum as they aged (Hwang et al., 2004; Zhang et al., 2004). Twenty-day old plants (with three fully expanded true leaves) are suitable to differentiate resistant and susceptible interactions as shown by Wang et al. (2000). However, the reported procedures for TRV-VIGS studies in Solanaceae mostly conduct agroinoculation on 14-days-old or older seedlings, and then perform with pathogen challenge several days afterward (Ekengren et al., 2003; Brigneti et al., 2004;
Bhattarai et al., 2007; Senthil-Kumar et al., 2007a).
Therefore, a cotyledon agroinfiltration method using 9 or 10-day-old seedlings were developed for our needs after evaluating several important factors. Systemic gene silencing effect was observed 5, 7 and 10 days post Agrobacterium infiltration in stembases, roots, and young leaves of tomato plants. In our established VIGS protocol, we challenged the silenced plants with R. solanacearum 10 days after agroinfiltration when systemic silencing effect has reached and the plants are about 20 days old. Currently, there are only two reports describing procedures applicable on younger seedlings: agrodrench (Ryu et al.,
2004) and vacuum infiltration (Hartl et al., 2008). Our
comparative study clearly revealed that agroinfiltration on cotyledons is much more efficient than agrodrench to trigger systemic gene silencing in young seedlings. Additionally, our preliminary experiments indicated that vacuum infiltration not only is much more time consuming, also would cause damage on tomato true leaves. The cotyledon agroinfiltration method established in this study would be applicable for the studies of other soil-born vascular pathogens.
Consistent with previous reports (Brigneti et al., 2004;
Senthil-Kumar et al., 2007b), VIGS efficiency of the
developed cotyledon agroinfiltration method varied among S olanaceous species and cultivars. All tested tomato cultivars were similarly susceptible to the method and efficiently revealed the effects of gene silencing (Table 2). However, only three of the seven tested pepper cultivars and none of the test eggplant cultivars displayed gene silencing results. It is possible that peppers and eggplants require a longer time to achieve full gene silencing than tomatoes, similar to the observation noted in some species of potatoes (Brigneti et al., 2004). Alternatively, this may reflect host preference of TRV and/or A. tumefaciens strains among solanaceous species (Burch-Smith et al.,
2004; Ghazala and Varrelmann 2007; Hartl et al., 2008),
in which the level of Agrobacterium infection and viral replication/movement in various plant species might vary and consequently affect VIGS efficiency. Besides, it is also plausible that the PDS sequences used did not have
TRV TRV-COM L390 TRV TRV-COI1
Figure 5. Silencing of COI1 resulted in increase of R. solanacearum proliferation in tomato H7996 plants. Resistant cultivar H7996 and susceptible cultivar L390 were agroinfiltrated with A. tumefaciens strain GV3101 containing pTRV1 plus pTRV2 containing COI1 genes (silenced plants), or plus pTRV2 empty vector (TRV-infected plants). (A) VIGS-mediated reduction of COI1 transcript level. cDNA was synthesized from total RNA isolated from pooled samples of stembases collected from two plants 15 days after agroinfiltration and used for PCR amplification. Amplification of the internal control Ubi3 was performed. Primers used for COI1-specific amplification are listed in Table 1. The experiment was performed twice with similar results. PCR amplification from cDNA from a single representative sample is presented. PCR cycles are indicated on the top of the sections. Lane M indicates DNA ladder and NC indicates negative control where RNA was used as template for PCR amplification in the absence of reverse transcriptase; (B) R. solanacearum population in L390 and H7996 infected with TRV or pTRV-COI1. Assessment was carried out 5 DPI by a method described in Materials and Methods. Numbers of positively detected plants over total assayed plants were indicated above each column. Means were calculated from data of all assayed plants and pair-wise mean comparisons were made between TRV-infected and silenced plants of H7996 with Student t-test method (*, significant at 0.05 level; **, significant at 0.01 level).
HO et al. 一 Optimizing TRV-VIGS system for systemic diseases 421
sufficient homology in other cultivars of peppers and eggplants. Additionally, manipulation of environmental conditions, such as temperatures for plant growth, might enable desired gene silencing effects in peppers and eggplants. Future studies would help clarify the causes for the low VIGS efficiency observed in peppers and eggplants, and lead to further optimization of the system in these plants.
As plant traits often are controlled by multiple genes and pathways acting redundantly or synergistically, simultaneous reduction of the expression of multiple genes would be necessary to reveal complicated mechanisms, such as tomato resistance to R. solanacearum. Previously, Chen et al. (2004) demonstrated the effectiveness of co-silencing of two genes using TRV containing a tandem construct in petunia. Their design was to co-silence a gene of unknown function linked to silencing of a gene with a visible phenotype. Our study is the first report demonstrating the co-silencing of three genes. We showed that a chimeric construct of PDS with the other two genes was as efficient as a single PDS fragment in silencing PDS in roots, stembases, and leaves. More tests constructing different gene combinations would be needed to determine the limit of the chimeric approach to co-silence three genes. We also demonstrated the apparent reduction of the gene silencing effect caused by inoculation of multiple Agrobacterium lines either carrying empty RNA2 vector or vectors containing additional targeted genes. This implied that more than one TRV vector could not be co-expressed in a plant cell. A similar observation has been made by Giritch et al. (2006), showing that two different TMV vectors failed to co-express in cells of Nicotiana benthamiana via agroinfiltration. Our accumulated studies have demonstrated that more than one defense signaling pathways contribute to the resistance to BW in tomato plants, and that these pathways may interact in an additive
mode (Lin et al., 2004; Chen et al., 2009). The multiple-
gene silencing protocol developed in this study could be used to clarify the interactions and contributing level of different signaling pathways.
Our study is the first report revealing that the tomato defense mechanism against BW involves the JA signaling pathway. Prior to this study, limited information was available, primarily on Arabidopsis. COI1 mutant of susceptible Arabidopsis Col0 ecotype displayed milder disease symptoms compared with the wild-type plant in response to R. solanacearum infection (Hernandez-Blanco et al., 2007), which suggested the JA signaling pathway may play a negative role in Arabidopsis defense to R. solanacearum. However, our results showed that silencing COI1 in the bacterial wilt-resistant tomato cultivar H7996 resulted in systemic increase of R. solanacearum proliferation, suggesting the JA signaling pathway is positively involved in tomato defense mechanism against this bacterium. Therefore, our findings provide additional support for differences in R. solanacearum interactions with tomato plants and Arabidopsis (Lin et al., 2008), although knocking down the target genes by taking a
transgenic RNAi approach would further help fortify these results.
In nature, plants orchestrate multiple defense signaling pathways to achieve proper response to various pathogen attacks (Pieterse and Dicke, 2007). In this study, we demonstrated the involvement of the JA signaling pathway in tomato defense against BW. Studies in tobacco have demonstrated that silencing players of mitogen-activated protein kinase (MAPK) cascades led to altered JA level, and thus resulted in changes of SA accumulation (Seo et al., 2007) and the JA-dependent signaling pathway (Kandoth et al., 2007). In Arabidopsis, MAPK cascades also are suggested to be involved in the ET signaling transduction pathway (Yoo et al., 2008). Additional results obtained from our VIGS assays on three SA-related candidate defense genes further suggested a positive role of SA related defense signaling pathways in tomato resistance to BW (Chen et al., 2009). Therefore, the protocol established in this study will be an effective tool to systemically evaluate the interplays of MAPK cascades, JA/ET and S A pathways in the tomato BW-defense network to R. solanacearum.
Acknowledgements. The authors thank Dr. Savithramma Dinesh-Kumar for providing the TRV vectors. Technical assistance by Ms. Chuang Mei-Hong and statistic advices by Ms. D. R. Ledesma are highly appreciated. This work was supported by grants from the Council of Agriculture, Republic of China (96AS-1.2.1-ST-a1-15 and 97AS-
1.2.1-ST-a1-17 to C. -P. Cheng and J. -F. Wang), and from National Taiwan University (97R0066-37 to C. -P. Cheng).
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利用煙草脆裂病毒基因靜默系統研究土壤傳播性維管束病原菌
Ralstonia solanacearum引起之病害
何芳儀1,3 陳永義2,3 林玉梅2 鄭秋萍2 王肇芬1
1亞蔬世界蔬菜中心
2國立台灣大學植物科學研究所與生命科學系
由Ralstonia solanacearum引起的茄科作物青枯病是一具全球重要性的土壤傳播性維管束病害，然
而茄科作物對青枯病菌防禦機制的研究及資料卻尚闕如。在此研究中，為了應用病毒誘導基因靜默系統
(VIGS) 於青枯病及其他土壤傳播性維管束病害相關研究，我們首先評估農桿菌菌株、植物株齡、接種
方法及茄科植物品種對phytoene desaturase (PDS)基因靜默效率的影響，將煙草脆裂病毒的VIGS系統最
佳化，並發展出子葉注射法。以此方法靜默PDS ，發現在所有供試番茄品種上均有良好靜默效果，而部
分番椒及所有茄子品種卻無明顯靜默效果。於9天大的番茄幼苗靜默植物抗病相關基因TGAL2.2的測試
發現，靜默效應在接種5天後已經在莖基部開始發生，接著在根部而後在幼葉呈現；同時將不同標的基
因之片段建構於同一載體中所達到之多基因靜默效果，比混合數個各自帶有不同標的基因片段農桿菌菌
株的方式更佳。利用本文建立之靜默系統進行茉莉酸防禦訊息傳遞途徑關鍵基因COI1之靜默，結果發
現青枯病菌在番茄抗病品種'Hawaii 7996'莖基部及中段內的菌量顯著增加，顯示茉莉酸防禦訊息傳遞
途徑在番茄對青枯病的防禦機制中扮演正面角色，此發現與阿拉伯芥對青枯病的防禦機制研究所知的相
反；文中並討論其他防禦訊息傳遞途徑可能如何共同參與番茄青枯病防禦網絡。
關鍵詞：青枯病；茉莉酸防禦訊息傳遞途徑；青枯病菌；病毒誘導基因靜默。