Enhanced Virus Resistance in Transgenic Maize Expressing a dsRNA-Specific Endoribonuclease Gene from E. coli
et al. (2013) Enhanced Virus Resistance in Transgenic Maize Expressing a dsRNA-Specific Endoribonuclease Gene from
E. coli. PLoS ONE 8(4): e60829. doi:10.1371/journal.pone.0060829
Enhanced Virus Resistance in Transgenic Maize Expressing a dsRNA-Specific Endoribonuclease Gene from E. coli
Xiuling Cao 0
Yingui Lu 0
Dianping Di 0
Zhiyan Zhang 0
He Liu 0
Lanzhi Tian 0
Aihong Zhang 0
Yanjing Zhang 0
Lindan Shi 0
Bihong Guo 0
Jin Xu 0
Xifei Duan 0
Xianbing Wang 0
Chenggui Han 0
Hongqin Miao 0
Jialin Yu 0
Dawei Li 0
Boris Alexander Vinatzer, Virginia Tech, United States of America
0 1 State Key Laboratory of Agro-Biotechnology and MOA Key Laboratory of Soil Microbiology, College of Biological Sciences, China Agricultural University , Beijing , P. R. China , 2 Plant Protection Institute, Hebei Academy of Agricultural and Forestry Sciences , Baoding , P. R. China
Maize rough dwarf disease (MRDD), caused by several Fijiviruses in the family Reoviridae, is a global disease that is responsible for substantial yield losses in maize. Although some maize germplasm have low levels of polygenic resistance to MRDD, highly resistant cultivated varieties are not available for agronomic field production in China. In this work, we have generated transgenic maize lines that constitutively express rnc70, a mutant E. coli dsRNA-specific endoribonuclease gene. Transgenic lines were propagated and screened under field conditions for 12 generations. During three years of evaluations, two transgenic lines and their progeny were challenged with Rice black-streaked dwarf virus (RBSDV), the causal agent of MRDD in China, and these plants exhibited reduced levels of disease severity. In two normal years of MRDD abundance, both lines were more resistant than non-transgenic plants. Even in the most serious MRDD year, six out of seven progeny from one line were resistant, whereas non-transgenic plants were highly susceptible. Molecular approaches in the T12 generation revealed that the rnc70 transgene was integrated and expressed stably in transgenic lines. Under artificial conditions permitting heavy virus inoculation, the T12 progeny of two highly resistant lines had a reduced incidence of MRDD and accumulation of RBSDV in infected plants. In addition, we confirmed that the RNC70 protein could bind directly to RBSDV dsRNA in vitro. Overall, our data show that RNC70-mediated resistance in transgenic maize can provide efficient protection against dsRNA virus infection.
Funding: This work was supported by the National Science & Technology Specific Projects of China (2011ZX08003-001 and 2009ZX08003-010B). The funders had
no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
. These authors contributed equally to this work.
Maize is a very important global resource for human food and
animal fodder, and is also a key bioenergy source. The world
production of maize was 844.4 million tonnes in 2010 and maize
provides the second highest level of production of all food and
agricultural commodities (FAO, http://faostat.fao.org/). In
China, maize yields were 208.12 million tonnes in 2012, and were
slightly more than rice (204.29 million tonnes), so maize is now our
number one grain crop (National Bureau of Statistics of China,
Bulletin on the National Grain Output of China in 2012).
However several diseases affect maize production, and in China,
maize rough dwarf disease (MRDD) complexes contribute
substantially to yield losses. The first report of MRDD in China
was in 1954 [
], and numerous reports have appeared in different
areas since the initial description. In the past few years, disastrous
losses caused by MRDD have occurred in most maize growing
districts of China. For example, in Shandong province in 2008, the
affected area comprised more than 733,000 hm2, where 59,100
hm2 had to be replanted with other crops and another 16,700 hm2
had complete yield losses [
Maize rough dwarf virus (MRDV), Mal de R´ıo Cuarto virus (MRCV)
and Rice black-streaked dwarf virus (RBSDV), which belong to the
genus Fijivirus in the family Reoviridae [
], each contribute to
MRDD syndromes in different maize growing regions. A MRDV
association with the MRDD was first noted in Italy [
], and the
virus is now known to be distributed broadly in maize growing
regions of Europe [
], South America [
] and East Asia [
respectively. In China, RBSDV was first found in MRDD maize
plants in 2000 [
], and subsequent studies have confirmed that the
etiological agent of MRDD in China is RBSDV, rather than
]. The RBSDV genome consists of 10
doublestranded RNA (dsRNA) segments [
], and the virus is obligately
transmitted by the small brown planthopper (Laodelphax striatellus),
in which it multiplies. RBSDV can infect several cereal crops
including rice, maize, wheat and sorghum [
], so a significant
disease reservoir exists in China.
RBSDV-infected maize exhibits severe growth abnormalities,
including plant dwarfing, dark leaf greening and a vein clearing
that often results in white streaks with small white enations on the
lower surfaces of leaves and sheaths. In the most severe cases,
infected plants fail to set seed or produce ears [
] (Figure 1).
Maize germplasm harbors different resistance responses to
MRDD under natural infection [
]. These studies have
shown that maize resistance to RBSDV is a quantitative
characteristic controlled by polygenes, each of which has minor
]. So far, a major dominant gene encoding RBSDV
resistant has not been identified genetically. Moreover, highly
resistant cultivated maize varieties of any sort that can be used to
control MRDD in the field are not available in the Chinese elite
heterotic groups [
]. Thus, the result is that all cultivated
maize varieties in agricultural production are rated as either
susceptible or highly susceptible. Because of these problems, it has
not been possible to breed new resistant lines by traditional
breeding. Therefore, current control measures rely on using
pesticides to kill vector insects, or altering the sowing time to
improve field management [
However, another environmentally safe and cost-effective
option for reducing crop losses is resistance mediated by
transgenes. Since the pathogen-derived resistance (PDR) concept
was first proposed [
], various transgenic approaches based on
viral genes and sequences have been applied to many plant species
]. Nowadays, three-quarters of virus-resistant transgenic
plants express viral coat protein (CP) genes, and the remaining
transgenic plants contain a large variety of other viral coding
sequences and non-coding sequences to confer resistance [
Nevertheless, these approaches have potential problems, because
the resistance generated is generally effective against only one kind
of virus or closely related viruses. In addition, viral RNA derived
resistance could possibly lead to selection of mutant viruses that
could evade transgenic resistance targeting. Apart from the
multitude of PDR approaches, a number of alternative strategies
for antiviral resistance in transgenic plants using various genetic
resources have been explored over the past two decades [
Amongst these, expression or induced expression of
dsRNAspecific ribonucleases has been shown to be successful [
Sano et al. generated transgenic potato lines expressing the
yeastderived dsRNA-specific ribonuclease gene (pac1), and found that
transgenic potato and progeny potato tubers could suppress Potato
spindle tuber viroid (PSTVd) accumulation during infection and that
the protein PAC1 could digest PSTVd in vitro [
]. Ishida et al.
came to the same conclusion with pac1 transgenic tobacco and
chrysanthemum, which also exhibited resistance to viroid and
virus infections [
]. Transgenic tobacco harboring the E. coli rnc
gene encoding an RNase III endoribonuclease and a mutant gene,
rnc70, were resistant to infection of several disparate RNA plant
viruses with divided genomes, but not against viruses with a
singlestranded RNA genomes [
]. In addition, wheat transformed with
rnc70 exhibited high levels of resistance to Barley stripe mosaic virus
(BSMV) infection [
Ribonuclease III (RNase III) represents a highly conserved
family of dsRNA-specific endoribonucleases that have important
roles in RNA processing, post-transcriptional gene expression
control and other processes initiated by dsRNA in both
prokaryotes and eukaryotes [
]. RNase III encoded by the
rnc gene in E. coli has the simplest protein structure in the family
and also is a well-studied family member. RNC70 is an RNase III
(E117K) mutant that has been proven to bind but not cleave
dsRNAs in vitro [
]. In this study, we demonstrate that two
RNC70 transgenic maize lines recovered after more than ten
generations of self-crossing and selection have high levels of
resistance to MRDD caused by RBSDV. This is the first report of
transgenic maize lines with effective resistance to RBSDV in the
Generation of Transgenic Maize Lines Carrying rnc and
RBSDV genomic RNA is composed of 10 double-stranded
RNAs that might be potential substrates for RNase III. To identify
anti-RBSDV activity of RNase III, two maize inbred lines (Z3 and
Z31 genotype), were transformed with pAMM2024 or
pAMM2025, in which the full-length E. coli RNase III cDNA
(rnc gene) and the E117K mutant (rnc70) were inserted downstream
of the rice actin promoter respectively (Figure 2A). Maize
genotypes have different responses to T-DNA delivery [
and we noted that the Z3 and Z31 genotypes were transformed at
efficiencies of 0.00% and 2.33% with pAMM2024, and 0.65%
and 1.62% with pAMM2025 respectively (Table 1). In all,
sixtyfour T0 transgenic lines were generated and confirmed by
PCRsouthern blot assays (Figure 2B and C). Among the T0 lines,
twenty-five lines contained the wild-type rnc gene, and thirty-nine
lines were transformed with the rnc70 mutant (Table 1). Compared
with the rnc70 lines, transgenic maize expressing the wild-type rnc
gene exhibited anomalous phenotypes, including stunted and slow
growth, leading to no progeny seeds in the T1 generation, these
results are consistent with previous studies in transgenic tobacco
Propagation and Screening of Transgenic Maize Lines in the Field
To obtain transgenic maize lines with high levels of resistance to
MRDD, 101 T2 PCR-positive transgenic maize lines were
challenged in the field. All of the transgenic maize lines were
maintained entirely by self-pollination within each transgenic line
in every generation. Because RBSDV, the cause of MRDD in
China, is transmitted by small brown planthoppers (Laodelphax
striatellus), the density of the planthoppers carrying virus is an
important factor affecting MRDD field incidence and spread. In
addition, temperature and humidity also contribute to disease
]. Keeping these factors in mind, we screened several
fields in which MRDD exhibited an unusually high disease
incidence (Figure 3) to evaluate resistance elicited by the
transgenic maize lines. To further maximize infection efficiencies,
the transgenic lines were planted at specific times to correlate the
most susceptible periods of maize development with the maximum
migration of planthoppers into the field plots [
Disease indices (DI) and MRDD symptoms were evaluated
during the milk stage of maize seed development. Disease scores
were defined as follows: 0 (no visible symptoms), 1 (4/5 of normal
healthy plant height, with white streaks throughout the veins of
upper leaves), 2 (2/3 of healthy plant height, with visible dwarfing,
combined with dark greening and white streak symptoms
throughout the plant), 3 (more extreme dwarfing; ,1/2 of healthy
plant height, delayed tassels that failed to develop pollen, and very
small cobs; and 4 (1/3 of healthy plant height, with no cobs or
dwarf cobs containing only a few seed, or plant death) (Figure 4).
Based on the disease score and the susceptibility rate, DI scores
were calculated and analysed [
] (Table 2). According to the DI,
the maize lines were divided into highly resistant (0.0–10.0),
resistant (10.1–20.0), susceptible (20.1–40.0) and highly susceptible
(40.1–100.0). The most highly resistant lines with positive genomic
PCR bands were selected for the next round of evaluation.
As shown in Table 2, 11.9%, 20.0% and 47.4% of the T2, T4
and T6 generation, respectively were highly resistant to MRDD.
In these experiments, transgenic maize was planted in the winter
in tropical field plots in southern China and in northern areas in
the summer to obtain two generations of seed per year. The
resistance of the T3 and T5 generations was not evaluated during
the winter in tropical field plots because of the low populations of
L. striatellus carrying RBSDV in the field and the lack of optimal
temperatures for RBSDV infection. In the T6 generation, all of
the highly resistant transgenic maize lines were progeny of the
ND60 and ND67 T2 generation transgenic lines, both of which
had been transformed Z31 inbred line with rnc70. These results
suggest that the rnc70 resistance in the transgenic lines was durable
and had a selective advantage over 5 generations (T2 to T6) of
seed increase during the field conditions present from 2006 to
The systematic PCR analyses conducted to detect the transgene
in the T6 generation revealed that all of the samples were positive
for rnc70. Moreover, only a single rnc70 band was detected in
southern blot analyses of the samples, indicating that rnc70 had
integrated only as a single copy gene. In addition, the ND60 and
ND67 progeny exhibited no obvious changes from the recipient
inbred line (Z31) in either yield potential or other agricultural
The ND60 and ND67 Transgenic Maize Lines Exhibit High
Field Resistance to MRDD
To investigate the performance of the highly resistant transgenic
maize lines under different disease stress conditions, we conducted
field trials of the ND60 and ND67 T6, T8 and T10 generations in
the summers of 2008, 2009 and 2010 (Table 3). Because the
outbreak of plant viruses induced diseases is affected by numerous
biotic and abiotic factors, we investigated MRDD resistance of
transgenic maize lines in several experimental field plots. To
provide a control for disease stress induced by RBSDV in the field,
we compared the transgenic lines with the cultivated maize
variety, Ye107, which is high susceptible to RBSDV.
In 2008 and 2009, Ye107 had normal susceptibility rates
(52.4% and 50.0%) and disease indices (52.4 and 32.5), indicating
that RBSDV field stress was relative low. Nonetheless, both of the
Measurements of disease score and disease indices are as in Figure 4 and Table 2. Values with different superscripts in the disease index column are significantly
different (P#0.05), as assessed by Duncan’s multiple range test.
**highly susceptible MRDD variety as a control.
T6 and T8 ND60 lines and ND67 lines exhibited high MRDD
resistance (DI 0.0–2.6, 0.0–4.1). In 2010, MRDD was prevalent in
the susceptible Ye107 control variety (97.2% and DI of 91.2), and
non-transgenic Z31 plants also exhibited high susceptibility rates
(72.6% and DI of 56.2). In contrast, six out of seven transgenic
ND67 lines in generation 10 (T10) were resistant, but two of the
ND60 progeny lines that were resistant in 2008 and 2009
exhibited the similar levels of susceptibility as the non-transgenic
Z31 plants (Table 3).
In each of the three stress years tested, all T6, T8 and T10
ND67 transgenic lines maintained stable levels of high MRDD
resistance compared with the non-transgenic plants (Figure 5),
and, except for 2010, most ND60 transgenic lines were also
remarkably resistant to disease development, whereas both lines
had better performance than the non-transgenic Z31 line (Table 3).
Among the progeny lines ND60 (ND60-2-5-4-1) and ND67
(ND67-1-3-5-5) consistently exhibited outstanding performance.
Overall, these results provide evidence that the rnc70 gene was
stably integrated into the transformed lines and was maintained
throughout several generations of variable disease and field
selection conditions, which also strongly suggest that introduction
of rnc70 provides resistance to RBSDV in the field.
RBSDV Resistance Following Challenge with a High
Population of Virulent Vectors
As described above, variations in the major biotic and abiotic
factors affecting the occurrence of RBSDV in the field during
2008, 2009 and 2010 created difficulties in evaluating in
transgenic resistance to MRDD. To more precisely control the
inoculation conditions and virus titer, we developed a modified
procedure, in which small brown planthoppers (L. striatellus) were
used to inoculate transgenic and control maize lines under
laboratory conditions. In these experiments, ND67-1-3-5-5,
ND60-2-5-4-1 T12 lines and Z31 recovered in 2011 were
challenged with virulent small brown planthoppers by allowing a
3 day inoculation access period (IAP) on plants to be tested. Each
maize line had three different replicates, and the experiments were
carried out twice. Under the IAP conditions, all tested maize lines
developed symptoms similar to those observed in the 2010 field
tests (Table 4). The non-transgenic Z31 plants and the transgenic
ND60-2-5-4-1 lines developed an intense MRDD phenotype (DI
was 74.0–92.5 and 90.3–96.3 respectively) that was much more
pronounced than that of the transgenic ND67-1-3-5-5 plants (DI
We also conducted ELISA and western blots assays to
quantitate the accumulation of RBSDV in diseased plants
(Figure 6A and B). Consistent with the observed MRDD
symptoms, the accumulation of RBSDV in the ND67-1-3-5-5
line was lower than in the ND60-2-5-4-1 and non-transgenic
maize lines. However, compared to the non-transgenic Z31 plants,
infected ND60-2-5-4-1 had a substantially lower accumulation of
Measurements of disease score and disease indices are as described in Figure 4 and Table 2. The numbers I, II and III represent three independent progeny selected from
lines tested in the previous generations. Values with different superscripts in the disease index column are significantly different (P#0.05) by Duncan’s multiple range
RBSDV virus in spite of developing similar severe disease
symptoms. This result suggests that expression of rnc70 in
transgenic maize not only depresses the incidence of MRDD in
transgenic plants exhibiting high levels of protection, but also
reduces the accumulation of RBSDV in plants that show lower
levels of MRDD losses. Therefore, even modest levels of RBSDV
control by rnc70 could contribute to lower MRDD field disease
indices by reducing virus acquisition during vector feeding.
RNC70 Interacts Directly with the RBSDV dsRNA Genome
Given that RNC is known to cleave dsRNA, and that RNC70
can bind dsRNA without cleavage, we predicted that transgenic
RNC70 plants might mediate resistance by binding to RBSDV
genomic dsRNA. To evaluate this notion, southern and western
blot assays were carried out with extracts from the transgenic
maize lines. The southern blot results confirmed that lines
ND671-3-5-5 and ND60-2-5-4-1 contain a single copy of the rnc70 gene
and express the full length RNC70 protein (Figure 7A and B).
Then, his-tagged RNC and RNC70 proteins were expressed in E.
coli and purified by metal affinity chromatography (Figure 7C) for
in vitro binding assays to determine whether the recombinant
proteins could bind and/or cleave dsRNA from the RBSDV
genome. His-tagged RNC and RNC70 proteins were incubated
with RBSDV genomic dsRNA, and then separated on 5% native
PAGE gels. As shown in Figure 7D, the dsRNA genome of
RBSDV was completely cleaved by RNC. In contrast, RNC70
reduced the migration rates of the dsRNAs in the gel and failed
cleave the dsRNAs. To our knowledge, this is the first report to
show that the E117K mutant (RNC70) participates in binding
viral dsRNA but is deficient in cleavage, and this provides some
evidence to explain the mechanism of resistance.
In this work, we have generated several transgenic maize lines
expressing RNC70. Among the lines, ND67-1-3-5-5 was highly
resistance in most challenge tests, and was more resistant than the
other tested transgenic line (ND60-2-5-4-1) or non-transgenic
plants (Tables 3 and 4). It should be noted that high levels of
resistance was observed under field conditions where a wide range
of complex biotic and abiotic factors could affect plant disease
resistance. Although screening for transgenic progeny resistant to
pathogen infections is normally performed under artificial
inoculation conditions, screening in the field, as undertaken in
our study, provides an efficient and economical means for
evaluating virus-resistant transgenic plants [
although the stable RNC70 expression levels in T12 transgenic
plants were similar (Figure 7B), resistance against RBSDV in the
ND67 lines was higher than those of the ND60 lines, both in the
field and under artificial inoculation conditions (Tables 3 and 4).
These differences could be due to copy number, insertions at
positions that affect transcription, different levels of transgene
silencing and epigenetic effects, each of which could cause
variations in the levels of protein expression and result in reduced
In addition to causing a major disease in maize, RBSDV can
infect wheat, rice and sorghum, where it can also cause serious
]. RBSDV invades rice and causes rice black streaked
dwarf disease, which is one of most serious viral diseases in rice in
China. Wheat and sorghum, infected with RBSDV also exhibit
dwarfing syndromes. RBSDV is obligately transmitted to each of
these cereal hosts by the small brown planthopper (L. striatellus)
. Because the major hosts of the small brown planthopper are
very important cereal crops that are grown throughout China, an
RBSDV epidemic in one crop could easily have effects on
contiguous crops and result in yield losses in all of the crops.
Continuous cropping or mixed cropping, as well as the presence of
weed hosts of the small brown planthopper throughout the entire
year also leads to persistent cycles of infection. For example, the
wheat-maize rotation areas of northern China are especially
hardhit by MRDD, and the main reason for this is planthopper
transmission of the virus from wheat to maize seedlings [
results suggest that expression of rnc70 in transgenic maize could
not only reduce MRDD losses, but could also affect RBSDV
reservoirs in maize field, thus decreasing the amount of viruliferous
planthoppers migrated subsequently into wheat, rice and other
In 2008, a new Fijivirus species, Southern rice black streaked dwarf
virus (SRBSDV), closely related to RBSDV, was reported in a rice
field in southern China [
]. Since then, SRBSDV has been
identified in rice fields in other provinces in China and in
Vietnam, and has even been diagnosed in a maize field in
northern China [
]. If SRBSDV spreads, it will be very
difficult to control, because the insect vector, the white-backed
planthopper (Sogatella furcifera), is a wide-spread pest that has an
extensive migration range in China and many other Asian
countries, and hence can be expected to effectively transmit the
virus from rice to maize crops [
]. We have verified that use of
RNase III-based transgenic maize is a viable strategy for RBSDV
control that likely can be applied to other transgenic crops to
prevent virus diseases, particularly those caused by dsRNA viruses.
For example, previous studies have shown that rnc70 transgenic
tobacco plants are resistant to infection by several disparate RNA
plant viruses with divided genomes [
], and wheat transformed
with rnc70 exhibits resistance to BSMV infection [
In summary, our study shows that transgenic maize plants
harboring rnc70 exhibit resistance to RBSDV and suggests that the
transgene likely will be effective against other phytoreoviruses.
Moreover, wide spread utilization of rnc70 resistance should
reduce transmissibility of RBSDV in the field and provide
protection to RBSDV host crops in the same area. Hence, in
addition to wheat and maize, we expect that rnc70 resistance will
be applicable to rice, sorghum and other field crops.
Materials and Methods
Construction of rnc Gene and its Mutant (rnc70)
The expression vectors, pAMM2024 and pAMM2025, a gift
from professor Amitava Mitra (Department of Plant Pathology,
University of Nebraska-Lincoln), contain the gene rnc from E. coli
(GenBank accession number X02946) and its E117K mutant
(rnc70), respectively. The backbone of those two plasmids is
pCB301, which is a mini binary transgenic vector that contains the
broad-host-range RK2 replication origin nptII for kanamycin
resistance and the T-DNA border sequences with the multiple
cloning site (MCS) polylinker from pBlueScript II [
681 bp ORFs of rnc and rnc70 were inserted into MCS sites
between the rice Actin 1 promoter and nopaline synthase (Nos)
transcription terminator (Figure 2A). The binary expression
plasmids containing the rnc or rnc70 genes were transferred to
Agrobacterium tumefaciens strain LBA4404 for maize transformation.
Maize Transformation and Identification
The maize inbred lines Z3 and Z31 belong to one of six Chinese
elite heterotic groups. These two lines have been used for
development of several hybrid progeny lines grown throughout
] and exhibit high genetic transformation efficiency and
are widely used as transgene recipients in China [
Transformation of Z3 and Z31 was achieved through an
Agrobacteriummediated approach as previously described [
]. Briefly, immature
zygotic embryos of the inbred maize lines were aseptically
dissected from ears harvested 10 to 13 days post pollination and
transformed in four different combinations using the expression
vectors (pAMM2024 and pAMM2025) and two inbred lines (Z3
and Z31). T0 plants were regenerated in tissue culture and selected
on 10 mg/L kanamycin media. The rnc and rnc70 genes were
identified in transformed plants by PCR with the pRN3-3
(59CAACGGAAGCTGGGCTACAC-39) primer corresponding to
28–47 nt of the rnc gene and the reverse pRN3-4 primer
(59TTGAACCTGTGCCAACCACC-39) complementary to 619–
600 nt of the rnc gene. The PCR products from the transgenic
plant genome DNA were transferred to nylon membranes,
followed by hybridization with the 32P-labeled probe from the
rnc70 gene to provide PCR-Southern blots.
For genomic southern blots, 30 mg of DNA from the T12
transgenic maize leaves was digested with Hind III and EcoR I
respectively. The digested DNA products were separated on a 1%
agarose gel, transferred to a nylon membrane and hybridised with
a 32P-labeled random primed fragments generated from the
681 bp rnc70 gene.
Purification of His-tagged RNC70 Proteins and Antibody
The rnc70 gene was amplified with the RNC70-1
(59-CCGCTCGAGTCATTCCAGCTCCAGTTTTTTC-39) primers. The BamH I and Xho I sites
(underlined), of the primers were used for introduction into the
prokaryotic expression vector pET-30a(+). The recombinant
vector was then transformed into E. coli strain BL21. One liter
bacterial cultures were grown to 0.6 OD600 from selected colonies,
induced with 1 mM IPTG, and shaken overnight at 28uC. The
induced cells were lysed by sonication and centrifuged at 4uC,
12000 rpm for 30 min. The cleared lysates were passed through
nickel resin (Qiagen China Co., Shanghai) and the His-tagged
fusion proteins were eluted with 100 mM imidazole and used to
elicit antibodies and for dsRNA binding. Fusion proteins used to
elicit rabbit antibodies were dialysed in 50 mM PBS buffer with
500 mM NaCl before intramuscular injections. Fusion proteins
used for dsRNA binding assays were dialysed against 20 mM
TrisHCl (pH 6.4), 50 mM NaCl, 1 mM EDTA, 0.5 mM DTT and
50% glycerol [
Western Blot and ELISA Assays
For western blots, 0.1 g of fresh leaf tissues were collected and
soluble proteins were extracted by grinding in 200 ml of protein
loading buffer, separated on 15% SDS-PAGE and transferred to
nitrocellulose filters. Primary antibodies against RNC70 were used
at a dilution of 1:500, and the enzyme-linked secondary goat
antirabbit antibody conjugated to alkaline phosphatase (AP-A) (Sigma
Aldrich, St. Louis, MO) was diluted to 1:5000. The AP-A
substrate, BCIP/NBT, was added, and the reaction was stopped
by washing out the substrate.
For ELISA, 50 mg of fresh maize leaves were extracted in
200 ml of 10 mM ammonium citrate buffer (pH 6.5) for evaluation
of total protein. Duplicate samples consisting of 80 ml of extraction
solution and 20 ml of citrate buffer were added to 96 well plates,
and incubated overnight at 4uC. The plates were washed with
0.02 M phosphate-buffered saline containing 0.05% Tween 20
(PBST) and then blocked with 3% BSA prepared in PBST for 2 h
in 37uC. After PBST washing, plates coated with bound antibodies
against the outer capsid (P10) of RBSDV for 2 h in 37uC [
Then, the PBST sample washing was repeated, and the secondary
AP-A antibody was added to the plates, incubated for 1 h in 37uC.
In the final step, plates were rinsed with PBST, incubated with
pnitrophenyl-phosphate (pNPP), and the OD405 was determined with a
spectrophotometer (Model 650, Bio-Rad).
Experimental Transmission of RBSDV to Maize by the
Small Brown Planthopper
Healthy L. striatellus second instar nymphs were placed on wheat
infected with RBSDV for 3 days of virus acquisition and feeding,
followed by transferring to healthy wheat. After 20 days, the
number of planthoppers containing RBSDV was assessed by
ELISA and shown to range from 23 to 27%. Then, 800
planthoppers were transferred to 210 maize seedings at 10 days
after planting, when the first two true leaves had fully expanded
and the leaf whorl was beginning to emerge. The plants were
placed in an insect-proof net cage (60 mesh), and after a 3-day
inoculation access period, the planthoppers were killed with an
insecticide. The maize plants were grown to the four-leaf stage,
and transplanted to an experimental plot covered with a fly net to
prevent external planthopper access. Leaves were collected for
molecular detection after 10 to 15 days when visible symptoms
appeared, and the plants were grown to maturity to assess the
RBSDV dsRNA Genome Isolation and Binding with RNC70
To isolate dsRNA, 5 g of fresh RBSDV infected maize leaves
were ground in liquid nitrogen, and 9 ml of 16STE (0.05 M
TrisHCl (pH 6.8), 0.1 M NaCl, 1 mM EDTA) and 1 ml 2% SDS was
added. An equal volume of water saturated phenol: chloroform:
isoamylalcohol (25:24:1) was added and stirred with a magnetic
stirrer for 30 min at room temperature. The samples were
centrifuged at 8000 rpm for 20 min, the supernatant was
recovered, and ethanol was added to a final concentration of
16%. The mixture was then passed through a 2.5 g cellulose CF11
affinity column and washed with 30 ml of 16 STE containing
16% ethanol followed by a second wash with 120 ml of 16 STE
containing 16% ethanol to remove residual ssRNA. The dsRNA
was eluted with 15 ml of 16 STE without alcohol and the eluent
was collected. RNA was precipitated by adding 1/10 volume of
3 M NaAc and 2.5 volumes of cold absolute ethanol, and the
samples were stored at 220uC for at least 2 h. Samples were then
centrifuged at 12000 rpm for 15 min washed twice with 70%
alcohol and the pellets were dissolved in ddH2O or 16 STE and
stored at 220uC.
RNA binding was carried out in reactions containing 75 ng of
purified RBSDV genomic dsRNA and 0.11, 0.22 or 0.33 mg of
his-tagged RNC70 protein in 10 mM Tris-HCl, 10 mM MgCl2,
50 mM NaCl, 1 mM dithiothreitol (pH 7.9). The reactants were
incubated for 30 min at 37uC, and electrophoresed on 5% native
polyacrylamide gels at 20 V for 4 h before ethidium bromide
staining and photography of the gels.
Resistance Evaluation and Statistical Analysis of
To evaluate the resistance of transgenic maize plants under field
conditions, transgenic plants and non-transgenic controls were
grown in areas with the highest disease incidences in the region
(Figure 3). Depending on the availability of planting material, two
to ten replicates of single-row plots per transgenic line were grown
using a randomized block design. Each row, consisting of ten to
twenty plants, was planted with seed from a randomized selected
ear of each transgenic maize line.
Every maize plant evaluated was assigned a disease score and
the numbers of plants corresponded to the numbers of plant lines
recommended by the SPSS analysis package described below. All
infectivity data were incorporated into Microsoft Excel 2010, and
the results were analyzed with SPSS Statistics software (version 17,
IBM). The statistical analyses were calculated using the disease
scores and the corresponding numbers of plants in each line were
analyzed by Duncan’s multiple range tests to evaluate statistical
differences between the resistances of each line.
We thank Professor Andrew O. Jackson (Department of Plant and
Microbial Biology, University of California at Berkeley) for helpful
suggestions and constructive criticism, Professor Amitava Mitra
(Department of Plant Pathology, University of Nebraska-Lincoln) kindly provided
the expression vectors pAMM2024 and pAMM2025.
Conceived and designed the experiments: DL JY HM. Performed the
experiments: XC YL DD ZZ HL LT AZ YZ LS BG JX XD. Analyzed the
data: DL HM JY XC YL DD CH XW. Wrote the paper: DL YL JY HM.
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