全 文 ：Received 1 Aug. 2003 Accepted 20 Sept. 2003
Supported by the Hi-Tech Research and Development (863) Program of China (2001AA222091), Knowledge Innovation Project of The
Chinese Academy of Sciences (KSCX2-1-01) and the National Special Program for Research and Industrialization of Transgenic Plants
(J00-A-002).
* Author for correspondence. E-mail: .
http://www.chineseplantscience.com
Acta Botanica Sinica
植 物 学 报 2004, 46 (6): 744-750
Isolation and Characterization of Mla-like Genes of Wheat
CAO Shuang-He, WAN Ping, ZHANG Xiang-Qi*
(State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology,
The Chinese Academy of Sciences, Beijing 100101, China)
Abstract: According to the conservative region of resistance gene homologue (RGH) family of the
barley Mla locus, we synthesized four pairs of family primers. RT-PCR with the series of family-specific
primers was used to analyze the differential expression of genes between the inoculated seedlings and the
uninoculated seedlings of a wheat (Triticum aestivum L.) line which showed high resistance to powdery
mildew. As a result, a putative resistance gene fragment, RJ-3-3L, expressed specifically in the inoculated
condition, was isolated and then, its full-length cDNA, designated as TaMla1, was obtained using rapid
amplification of cDNA ends (RACE). By alignment analysis and functional-motif scanning, the TaMla1 shared
high similarity to Mla alleles in barley and coded a typical CC-NBS-LRR (coiled-coil, nucleotide binding site,
Leu-rich repeat) resistance protein. Subsequently, by Southern blot using a series of nulli-tetrasomics of
wheat, the TaMla1 was located on wheat chromosome 1A, where the ortholog of the barley Mla locus lies.
These findings showed that TaMla1 is one of the Mla-like alleles in wheat. In addition, another gene
fragment, RW-2-3L, its transcription remarkably inhibited in the inoculated seedlings, was isolated and
homology analysis showed that it was also highly similar to Mla members in barley. We inferred that the
gene derived RW-2-3L was also a Mla-like allele and probably acted as a powdery mildew susceptible gene or
a negative factor for resistance to powdery mildew.
Key words: wheat; Mla-like gene; resistance to powdery mildew; negative or susceptible factor
Powdery mildew fungus is one of the most widespread
plant pathogens and infects thousands of dicotyledon and
monocotyledon including many important crops, such as
wheat, barley, rice, and tomato, and the losses in plant
growth and yield attributable to this disease are probably
greater than any others (Nancy, 2002). At present, quite a
little breakthrough was accomplished on the study of pow-
dery mildew of barley and Arabidopsis. However, the study
on this aspect of wheat, which is one of the two most se-
verely affected crops by powdery mildew, still retained on
the exploitation of its resistance resource, identification and
location of resistance genes because of its large genome
and hexaploidy. Delightfully, several resistance genes have
been isolated from barley Mla and Mlo loci conferring race-
specific resistance and broad-spectrum resistance to pow-
dery mildew, respectively, and the homological locus in
wheat (designated as TaMla, TaMlo1A, TaMlo1B and
TaMlo1D) have been identified by comparative genomics
method, which supplied predominant condition for isolat-
ing the Mla-like and Mlo-like resistance genes of wheat
(Buschges et al., 1997; Elliott et al., 2002; Wei et al., 2002).
In addition, an OsMlo gene of rice had been cloned using a
protein-protein interaction-based screening of a cDNA ex-
pression library constructed from pathogen-elicited sus-
pension cell line just according to barley Mlo protein prop-
erty (Kim et al., 2002).
The barley Mla locus is a gene cluster including 32
alleles. Fifteen of them are associated with plant defense
response and are grouped into five families including three
CC-NBS-LRR resistance gene homologues (designated as
RGH1, RGH2 and RGH3), one chemically induced family
and one Cl2 family (Wei et al., 2002). At present, of the
isolated alleles from Mla locus, Mla1, Mla6 and Mla13
have been verified to be resistant to powdery mildew using
single-cell expression assay and they specifically recog-
nized different Avir genes of pathogens and had different
requirement for the downstream signaling components, Rar1
or SGT1 (Halterman et al., 2001; Zhou et al., 2001; Azevvedo
et al., 2002; Halterman et al., 2003). This implied that they
accomplished their resistant function through different sig-
naling systems or/and reflected grossly different temporal
or spatial Avir gene activities during pathogen invasion.
CAO Shuang-He et al.: Isolation and Characterization of Mla-like Genes of Wheat 745
However, Mla1, Mla6 and Mla13 shared high similarity
whether at the nucleotide or amino acid level, which pro-
vided indispensable condition for effectively isolating the
Mla-like gene of wheat using PCR with family-special prim-
ers (Elliott et al., 2002). In addition, all of the three Mla
alleles conferred a rapid defense response phenotype by
inhibiting the haustorium differentiation of powdery mil-
dew pathogen, which has predominant value in use com-
paring to the delayed and intermediate response conferred
by other genes in the Mla locus (Wei et al., 2002). However,
they need different signal components to function.
Obviously, even if resistance genes among the relative
species shared high similarity in structure, each of them
had own genus-specific manner. Thus, it was not only fea-
sible but also necessary to isolate the wheat homologues
of barley Mla genes. In addition, it was helpful not only to
improve the understanding of resistance to powdery mil-
dew but also to supply a lot of information for comparative
genomic research and genetic evolution (Wei et al., 2002).
In this paper, the gene differential expression strategy
based on RT-PCR technique was employed to isolate Mla-
like gene from a wheat line that is resistant to powdery
mildew. As a result, a full-length putative resistance to pow-
dery mildew gene was isolated and located on the wheat
homologue of the barley chromosome containing Mla
locus.
1 Materials and Methods
1.1 Materials and inoculation treatment
Wheat-rye 6BS/6RL translocation line TAM104R show-
ing excellent resistance to quite a lot of Bgt isolates (Friebe
et al., 1994) was used to isolate Mla-like genes. The
TAM104R line was kindly provided by Dr. Friebe.
TAM104R seedlings were inoculated by E18, a prevalent
powdery mildew isolate with broad virulence to wheat
(DUAN Xia-Yu, personal communication). The same tis-
sues were harvested from both inoculated and
uninoculated seedlings at 5 h, 10 h, 15 h, 20 h, 23 h, 29 h
and 48 h after inoculation and snap-frozen in liquid
nitrogen. The E18 isolate was kindly provided by Profes-
sor DUAN Xia-Yu and inoculation experiment was carried
out in Institute of Plant Protection, The Chinese Academy
of Agricultural Sciences.
1.2 Total RNA isolation and reverse transcription
Total RNA was isolated using GT (Guanidine
Thiocyanate) method described as Sambrook et al. (1989).
Subsequently, reverse transcription using total RNA as
template was performed to produce cDNA and its proce-
dure was just described as the protocol (M-MLV) of
Promaga.
1.3 Primer design and RT-PCR
Four pairs of family-specific primers, P1, (1LF: 5-
AT GGATAT T GT CACCGGT GCC-3 ; 1 LR: 5 -
TCAGTTCTCCTCCCTCGTCC-3). P2, (2LF: 5-
GATTGTCGGG(C/T)(T/A)TTT GTTCCG -3; 2LR: 5-
ATGTA(G/A)GTTGCACAGAGAC-3). P3, (3LF: 5-CCTT
(G/T)CCTT(C/A)GAGCTTTGTAT-3 ; 3LR: 5 -
GCTTCCTTTGCCTCCCC(A/C)AC-3) and P4, (4LF: 5-
CCACA ACCCGCATTGTCAGTG-3; 4LR:5-C(T/A)
GTGGACGGCAATTC(C/A) TT-3), were designed accord-
ing to different conserved region of RGH gene family from
barley Mla locus and synthesized by Augct, Beijing,
In a total volume of 20 µL, the RT-PCR mixture con-
tained 2 µL reverse transcription product, 1× buffer and
0.5 U ExTaq polymerase (TaKaRa, Dalian), 200 pmol /L
primers, and 100 µmol/ L dNTPs. The PCR profile was just
as follows: 94 ℃ for 4 min for denaturing, followed by 35
high-stringency cycles comprising of 94 ℃ for 45 s, 60 ℃
for 2 min and 72 ℃ for 1 min, and finally 72 ℃ for 5 min for
extension.
1.4 Electrophoresis, recovery and Northern blot hybrid-
ization
RT-PCR products were separated on 1% agarose gel
and displayed with EtBr staining. The target bands of inter-
est were excised using Gel Extraction Kit (Omega). The re-
covery procedures were referred to its technical booklet.
The total RNAs extracted from inoculated and uninoculated
leaves were fractionated in 1.2% agarose-formaldehyde gel
and then blotted onto Hybond-N+ membrane. Hybridiza-
tion was performed using [a-32p]-dCTP-labeled target gene
fragments as probe (Sambrook et al., 1989).
1.5 Cloning and sequencing
Recovery DNA fragments and pGEMâ-T vectors
(Promaga) were ligated together using T4 ligase and then
transformed into the competent DH10B cells of Escheri-
chia coli. The clones were identified through blue/white
screening. In addition, PCR amplification using the corre-
sponding primers and M13 sequencing primers confirmed
the size of the insert. The positive clones were sequenced
by TaKaRa using ABI PRISM BigDyeTM Terminator Cycle
Sequencing Reaction Kit.
1.6 Rapid amplification of cDNA ends (RACE)
3 and 5 ends of the target gene fragment were obtained
by RACE using 3-Full RACE Core Set (TaKaRa) and
SMARTTM RACE (Clontech, USA), respectively, and the
elaborate procedures were just according to their user
manual. The recovering, cloning and sequencing in the
course of RACE were the same as above.
Acta Botanica Sinica 植物学报 Vol.46 No.6 2004746
1.7 Chromosome location
A series of wheat nulli-tetrasomic lines were used to
determine the chromosomal location of the cloned gene.
Their genomic DNAs digested by restriction enzymes were
hybridized to target gene fragment labeled by isotope a-
32P as probe. The procedure of Southern blot hybridization
was according to Sambrook et al. (1989).
2 Results
2.1 RT-PCR display
The first-strand cDNA synthesis from the total RNA
was primed by an oligo(dT) anchor primer. RT-PCR using
the first-strand cDNA as template was performed with the
RGH family primers. As a result, only one of the four pairs
of RGH family primers, 3LF and 3LR, was effective to ob-
tain PCR products, so this showed that RGH family primers
possess selectivity. As illustrated in Fig.1, there were sev-
eral gene fragments (just as pointed with the arrows in
Fig.1) showing differential expression between the inocu-
lated and the uninoculated seedlings, we suspected that they
were related to resistance to powdery mildew isolate E18.
2.2 Recovery, Northern analysis, cloning and sequenc-
ing
According to the location of family primers in Mla
genes, the amplifying fragment length of the target gene
should be about 2 kb. For the above-mentioned reason, we
recovered two amplification bands, RJ-3-3L and RW-2-3L,
with about 2 kb length (pointed with the arrows in Fig.1)
using Gel Extraction Kit (Omega E.Z.N.A) from PCR
products. Subsequently, the two target gene fragments
were used to hybridize with the total RNAs from the inocu-
lated leaves (RJ) and the uninoculated leaves (RW) at 20 h
after infection with E18, respectively. Both of them showed
differential transcription between the inoculated and the
uninoculated conditions (Fig.2A, B). In addition, Northern
analysis showed that the transcription of RJ-3-3L was par-
ticularly induced by E18 infection but the RW-2-3L was
Fig.1. Differential expression display between inoculated seed-
lings at 20 h after inoculation with E18 and uninoculated seedlings.
M, marker; RJ, TAM104R incubated by E18; RW, TAM104R
uninoculated. The arrows indicate the location of the target bands.
Fig.2. Northern blot hybridization. A. Total RNAs from the
inoculated leaves (RJ) and the uninoculated leaves (RW) at 20 h
after infection with E18 hybridized to RW-2-3L. B. RJ and RW at
20 h after infection with E18 hybridized to RJ-3-3L. C. Total
RNAs from the inoculated leaves (above) and uninoculated leaves
(middle) at different time after infection with E18 together with
16S rRNA and 28S rRNA (below) as a positive control hybrid-
ized to RW-2-3L, respectively. D. Total RNAs from the inocu-
lated leaves (above) and uninoculated leaves (below) at different
time after infection with E18 together with 16S rRNA and 28S
rRNA (below) as a positive control hybridized to RJ-3-3L,
respectively.
CAO Shuang-He et al.: Isolation and Characterization of Mla-like Genes of Wheat 747
remarkably inhibited under the same condition (Fig.2C, D).
The two target gene fragments, RW-2-3L and RJ-3-3L, were
cloned into pGEMâ-T vector and the positive clones were
sequenced in Bioasia (Shanghai). Homology search showed
Fig.3. Amino acid sequence alignment of TaMla1, RW-2-3L, Mla1, Mla6 and other two Mla-RGH1 family members. Shaded boxes
indicate similar residues. Conserved motifs within the NBS region are indicated above the sequence. Asterisks denote the putative solvent
exposed residues of the LRR region. RGH1e and RGH1f gene sequences differ by only one nucleotide, which does not cause any amino
acid change. The accession numbers of Mla1, Mla6 and three Mla-RGH1 family members are AY009938, AJ302295, AJ302296,
AJ302297 and AJ302298 in GenBank, respectively.
Acta Botanica Sinica 植物学报 Vol.46 No.6 2004748
that the two cloned fragments were highly similar to RGH
family members of Mla locus (data not shown) (http://www.
ncbi.nlm.nih.gov/ blast.html).
2.3 The full-length gene isolation and amino acid se-
quence alignment
The full-length gene of RJ-3-3L (2 108 bp), designed as
TaMla1, was obtained using RACE approach and its ac-
cession number in GenBank is AY222610. Its nucleotide
sequence was 2 813 bp and deduced amino acid sequence
has 876 amino acid residues. Subsequently, the amino acid
sequence alignment with known RGH members was per-
formed (http://www.ebi.ac.uk/clustalw/index.htm) and its
functional region was analyzed (http://pfam.wustl.edu/
hmmsearch.shtml) (Fig.3). The results indicated that TaMla1
was high homological to RGH members from barley Mla
locus, especially to Mla1 and Mla6 (90% and 88% identity,
respectively). As illustrated in Fig.3, TaMla1 protein also
contains five conserved motifs indicating a nucleotide-bind-
ing site, just like RGH1 and the kinase-1a (P-loop), kinase-
2a, kinase-3a, and conserved domain 2 motifs are all highly
similar to those of other NBS-LRR resistance proteins.
However, its conserved NBS domain 3 lacks the conserved
phenylalanine found in other resistance proteins contain-
ing NBS. The C-terminal region of the protein contains
eleven imperfect leucine-rich repeats (LRRs) with an aver-
age size of 26 amino acids. These LRRs conform to the
consensus motif LxxLxxLxxLxLxx(N/C/T)x(x)L observed
in other cytoplasmic R gene products (Jones et al., 1997).
Therefore, the TaMla1 belongs to CC-NBS-LRR protein. In
addition, the amino acid sequence deduced from RW-2-3L
(2 067 bp, GenBank accession number AF538040) was also
highly homologous to the RGH members (Fig.3), suggest-
ing that the RW-2-3L encoded an incomplete CC-NBS-LRR
protein related to powdery mildew disease. Thus, we in-
ferred that it resulted from selection for divergence of TaMla
locus.
2.4 Chromosome location
Chromosome location of TaMla1 was performed using
a series of nulli-tetrasomic wheat lines. These nulli-
tetrasomics genomic DNA digested by HamⅢ was hy-
bridized using TaMla1 as probe. The result indicated that
only nulli-1A line lacked one hybridized band that presented
in other nulli-tetrasomic lines (Fig.4). Based on this result,
it could be inferred that the TaMla1 gene lay on chromo-
some 1A of wheat.
3 Discussion
Previous studies have located the homological locus of
Mla onto chromosome 1A of wheat by Southern blot hy-
bridization and this locus was 0.7 cM from a resistance to
powdery mildew gene Pm3b (Wei et al., 2002). TaMla1 gene
isolated in this study was just located on chromosome 1A
of wheat and both of its nucleotide sequence and encoded
amino acid sequence were highly similar to Mla of barley.
In addition, the structural analysis of the protein deduced
from TaMla1 indicated that TaMla1 belonged to the CC-
NBS-LRR protein the most familiar. Thus, we judge that
TaMla1 is an Mla-like allele of wheat. However, more ex-
perimental evidence is needed to prove the resistance func-
tions of TaMla1 gene. To date, the function of several genes
from Mla locus of barley have been testified using single-
cell transient expression (Mla1 and Mla6) and succeeded
expression in wheat leaf cells (Mla6) (Halterman et al., 2001).
No doubt, the method identifying function of barley Mla
genes is also feasible to verify the function of the candi-
date resistance gene TaMla1.
Resistance and susceptibility are opposite sides of the
same coin, and research on disease resistance cannot be
conducted with reference to susceptibility (Nancy, 2002).
At present, quite a lot of negative and susceptible factors
were isolated through genetic analysis for susceptible plant
to disease, such as PMR6, MLo and EDR1 (Frye and Innes,
Fig.4. Southern blot hybridization of nulli-tetrasomic lines of wheat using TaMla1 as probe. CS, Chinese Spring; N1A-N1D, nulli-
tetrasomic lines; R, TAMR104R. The arrow shows the location of the target band.
CAO Shuang-He et al.: Isolation and Characterization of Mla-like Genes of Wheat 749
(Managing editor: ZHAO Li-Hui)
1998; Devoto et al., 1999; Vogel et al., 2002). Noticeably,
comparative sequence analysis now indicated that
RGH1abc is a susceptible allele of Mla locus from a barley
cv. Morex and shared the highest nucleotide and amino
acid similarity with the Mla1, Mla6 and Mla13 (Wei et al.,
1999). Interestingly, the RW-2-3L was expressed specifi-
cally under the uninoculated condition. In addition, the
nucleotide alignment analysis showed that RW-2-3L was
highly similar to RJ-3-3L and RGH family members of Mla
locus. In view of above-mentioned facts, we infer that
though the RW-2-3L probably originated from selection for
divergence of TaMla locus, it acted as powdery mildew
disease susceptible gene or a negative factor for resistance
to powdery mildew of wheat.
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