Download SHR5: a novel plant receptor kinase involved in plant–N2

Journal of Experimental Botany, Vol. 57, No. 3, pp. 559–569, 2006
doi:10.1093/jxb/erj041 Advance Access publication 5 January, 2006
RESEARCH PAPER
SHR5: a novel plant receptor kinase involved in
plant–N2-fixing endophytic bacteria association
F. Vinagre1,2, C. Vargas1,2, K. Schwarcz1,2, J. Cavalcante1,2, E. M. Nogueira1,2, J. I. Baldani3,
P. C. G. Ferreira1,2 and A. S. Hemerly1,2,*
1
Instituto de Bioquı´mica Médica, CCS, Universidade Federal do Rio de Janeiro, 21941-590 Rio de Janeiro, RJ, Brazil
2
Laboratório de Biologia Molecular de Plantas, Instituto de Pesquisas do Jardim Botânico do Rio de Janeiro,
Rua Pacheco Leão 915, 22460-030 Rio de Janeiro, RJ, Brazil
3
CNPAB/EMBRAPA, BR465, Km47 23851-970 Seropédica, RJ, Brazil
Received 3 June 2005; Accepted 31 October 2005
Abstract
Endophytic nitrogen-fixing bacteria have been isolated
from graminaceous plants such as maize, rice, and
sugarcane. They are thought to promote plant growth,
not only by fixing nitrogen, but also by the production
of plant hormones. The molecular mechanisms involved in this interaction are not yet clear. In this work,
the identification of a receptor-like kinase (RLK), named
SHR5, which may participate in signal transduction
involved in the establishment of plant–endophytic
bacteria interaction is described for the first time.
SHR5 seems to be part of a novel subclass of RLKs
present in a wide range of plant species. The expression
of this gene is down-regulated in sugarcane plants
associated exclusively with beneficial endophytic
bacteria and is not a general response caused by
micro-organisms or abiotic stress. In addition, more
successful sugarcane–endophytic bacteria associations have a more pronounced decrease in SHR5
expression, suggesting that SHR5 mRNA levels in plant
cells are inversely related to the efficiency of the
association.
Key words: Biological nitrogen fixation, diazotrophic endophytic
bacteria, gene expression, LRR-RLK receptors, plant–microbe
interaction, signalling, sugarcane.
Introduction
High levels of biological nitrogen fixation (BNF) contribution have already been described in some graminaceous
plants (Boddey and Döbereiner, 1995), including sugarcane
(Saccharum spp) (Urquiaga et al., 1992). Nitrogen-fixing
bacteria have been isolated from sugarcane tissues, including Gluconacetobacter diazotrophicus, Herbaspirillum spp, Azospirillum spp, and Burkholderia tropicae
(Cavalcante and Döbereiner, 1988; Baldani et al., 1992,
1996, 1997; Reis et al., 2004). Unlike rhizobium/leguminosae symbiosis, where bacteria are restricted to nodules,
these diazotrophs are endophytic, colonizing intercellular
spaces and vascular tissues of most plant organs without
causing damage to the host (James and Olivares, 1998;
Reinhold-Hurek and Hurek, 1998). They promote plant
growth possibly by fixing nitrogen and also by the
production of plant hormones (Sevilla et al., 2001). It is
not yet clear which mechanisms are involved in the
establishment of this particular type of endophytic interaction. The fact that distinct sugarcane genotypes have
different rates of biological nitrogen fixation (BNF)
suggests that plant genetic factors might be controlling
the process of bacteria recognition, colonization, and/or
nitrogen fixation (Urquiaga et al., 1992). Recently, ESTs
that are preferentially or exclusively represented in cDNA
libraries were identified from plants inoculated with G.
diazotrophicus or H. rubrisubalbicans, suggesting that the
plant is actively involved in the interaction (Nogueira et al.,
2001). Several of the genes identified are possibly involved
in different processes of plant/bacteria signalling (Vargas
et al., 2003).
Plants have a large family of receptor-like kinases
(RLKs) that have been implicated in mechanisms of perception and transduction of extracellular signals into the
cell (reviewed by Shiu and Bleecker, 2001a). Phylogenetic
* To whom correspondence should be addressed. E-mail: [email protected]
Published by Oxford University Press [2006] on behalf of the Society for Experimental Biology.
560 Vinagre et al.
analysis of the RLKs in A. thaliana revealed that more than
400 genes encode putative plant receptor kinases, defined
as proteins with a signal sequence, an amino-terminal domain with a single transmembrane region, and a carboxylterminal cytoplasmic kinase domain (Shiu and Bleecker,
2001b). There are several classes of plant RLKs, distinguished according to their extracellular domains, which can
potentially bind an array of molecules (Shiu and Bleecker,
2001a). The largest plant RLK class is characterized by
the leucine-rich repeats (LRR) motif in the ectodomain.
Nevertheless, out of the 216 LRR-RLKs in A. thaliana,
only 10 or so have known functions, and only four have
been extensively studied (Diévart and Clark, 2004).
Members of this plant subfamily were known to play roles
in diverse processes related to plant growth/development,
stress, defence against pathogens, and symbiosis (Shiu
et al., 2004).
In this study, the identification of a novel sugarcane
gene, named SHR5, involved in the association with
endophytic nitrogen-fixing bacteria is described. Sequence
analyses suggest that the SHR5 gene encodes a protein that
belongs to a subclass of the LRR-RLK protein family with
a biological function not yet described and present in a wide
range of different species. Gene expression studies show
that SHR5 expression is drastically reduced in plants
associated with the diazotrophic endophytes. They also
suggest that it is not a general stress response to microorganisms, but that it seems to be specific for beneficial
associations. In addition, the results indicate that SHR5
mRNA levels in plant cells are related to the efficiency
of the association between sugarcane and endophytic bacteria. As far as is known, this study is the first report on a
plant RLK that is involved in the association between plants
and endophytic N2-fixing bacteria.
Materials and methods
In vitro plant growth and micro-organism treatments
Sugarcane plantlets free of micro-organisms were obtained by sterile
meristem culture and micropropagated according to the method of
Hendre et al. (1983). In vitro-grown SP70-1143 rooted sugarcane
plantlets were inoculated as described by James et al. (1994) using
0.1 ml of 106–107 bacterial suspension. Controls were inoculated
with medium only. Seven days after the inoculation, plants were
harvested and examined for bacterial colonization by the Most
Probable Number (MPN) estimation, according to the methods of
Reis et al. (1994). All plants were maintained at 30 8C with an
irradiance of 60 lmol photons mÿ2 sÿ1 for 12 h dÿ1. The sugarcane
genotypes used in this work were: SP70-1143 (high inputs of N from
BNF), Chunee (low inputs of N from BNF), B-4362 (susceptible to
H. rubrisubalbicans), SP70-3370 (susceptible to Leifsonia xyli
subsp. xyli), and SP71-799 (susceptible to Puccinia melanocephala).
The endophytic diazotrophic bacteria used were Gluconacetobacter
diazotrophicus (PAL5 strain), Herbaspirillum seropedicae (HRC54
strain), H. seropedicae nifÿ (M2 strain, unable to fix nitrogen),
H. rubrisubalbicans (HCC103 strain), and Azospirillum brasilensis
(Sp245 strain). Inoculations with Agrobacterium tumefaciens (A281
strain) and Leifsonia xyli subsp. xyli (CTC B07 strain) bacteria were
performed with in vitro-grown SP70-1143 and SP70-3370 varieties,
respectively. In vitro-grown sugarcane plantlets (SP71-799 variety)
free of micro-organisms were transferred to a greenhouse, then
inoculated 15 d later with approximately 106 spores solution of
Puccinia melanocephala. The material was harvested 15 d later, with
the inoculated plants showing rust disease symptoms. Stalks of fieldgrown sugarcane SP86-155 and SP80-1842 varieties with and
without mosaic virus disease and leaf scald disease, respectively,
were germinated in a greenhouse and shoots were harvested 15 d
later.
Other plant treatments
In vitro-grown SP70-1143 rooted sugarcane plantlets, free of microorganisms, were subjected to different treatments: (i) heat shock
treatment at 45 8C or 50 8C for 15 min; (ii) saline stress treatment for
16 h by the addition of 1% or 2% NaCl to the medium; and (iii) auxin
treatment for 7 d by the addition of 2.42 lg mlÿ1 of indole acetic acid
(IAA) or naphthalene acetic acid (NAA) to the medium. All plants
were maintained at 30 8C with an irradiance of 60 lmol photons mÿ2
sÿ1 for 12 h dÿ1.
RNA extraction and cDNA synthesis
For each expression analysis experiment, three to five plantlets were
pooled and total RNA was extracted according to Logeman et al.,
(1987). First-strand cDNA was prepared by reverse transcription of 5
lg of DNase I-treated RNA using the ‘First-Strand cDNA Synthesis
Pharmacia Kit’ and Not-dT as primer, according to the manufacturer’s instructions. For cDNA-AFLP experiments, PolyA+ RNA was
isolated using Dynabeads Oligo (dT)25 mRNA Purification kit
according to the manufacturer’s instructions (Dynal., Hamburg,
Germany).
cDNA-AFLP
cDNA amplified fragment length polymorphism (cDNA-AFLP)
analysis was performed according to Bachem et al. (1996) with
modifications. Double-stranded cDNA was digested with the restriction enzymes SacI and MseI (New England Biolabs). Sequences
of primers and adaptors used for AFLP reactions were: SacI adapters,
59-CTCGTAGACTGCTACAAGCT-39/39-CATCTGACGCATGT59; MseI adapters, 59-GACGATGAGTCCTGAG-39/39-TACTCAGGACTCAT-59; SacI pre-amplification primer, 59-CTCGTAGACTGCGTACAAG-39; MseI pre-amplification primer, 59-GACGATGAGTCCTGAGTAA-39; SacI selective amplification primer, 59-GACTGCGTACAAGCTC+NN-39; and MseI selective amplification
primer, 59-GATGAGTCCTGAGTAA+NN-39. The selective SacI
primers were end-labelled using [c-33P]dATP. The pre-amplification
PCR conditions were: 30 s at 94 8C, 1 min at 60 8C, and 1 min at 72
8C (28 cycles). The selective touch-down PCR conditions were: 30 s
at 94 8C, 1 min at 65 8C (–0.7 8C cycleÿ1), and 1 min at 72 8C (13
cycles), 30 s at 94 8C, 1 min at 56 8C, and 1 min at 72 8C (18 cycles).
Selective amplification products were separated on 5% polyacrylamide gel run at 55 W and 50 8C until bromphenol blue dye reached
the end. Gels were dried onto 3MM Whatman paper (Whatman,
Maidstone, UK) and positionally marked before exposing to Kodak
Biomax MR film for 24 h. The bands of interest were cut from the gel
with a surgical blade, eluted, and re-amplified with the preamplification primers. The re-amplified cDNAs were subcloned
using the pGEM-T vector system (Promega, Madison, USA) and at
least three individual clones were sequenced using automated
sequencing. The sequences were included for further analysis only
when they were identical. Database searches were performed at the
NCBI World Wide Web server using the Basic Local Alignment
Search Tool (BLAST) network service (Altschul et al., 1997). Each
transcript-derived fragment (TDF) sequence was compared against
RLK in plant–endophytic bacteria association
all sequences in the non-redundant database using the BLASTX
program, in the EST database using the BLASTN program and in the
S. officinarum database of TIGR Gene Indices using the BLASTN
program.
SHR5 sequence analysis
39-Nested PCR was performed to clone the 39 end of the SHR5
cDNA. First strand cDNA was prepared from micro-organism-free
sugarcane plantlets as described above. cDNA was used as a template
for first-round PCR with an oligo dT anchor primer and a 59 SHR5
gene-specific primer. Pfx Taq polymerase (Invitrogen) was used in
the following cycling protocol: incubation at 94 8C for 5 min
followed by 30 cycles of 94 8C for 1 min, at 60 8C for 1 min, and
at 68 8C for 3 min, and finished at 68 8C for 5 min. Five ll of the firstround reaction was used in a second reaction with a second genespecific primer designed to anneal 39 to the first gene-specific primer
under the same PCR conditions. TDF gene-specific primers were
based on cDNA-AFLP 179 bp identified sequence. Primers used for
the first-round PCR were TDF5a 59-ACTTGAAGATCCTCTGGGCAT-39 and NotI dT 59-TGGAAGAATTCGCGGCCGCAGGAAT(18)-39, and for the second-round PCR: TDF5b 59-GCCTGACTCAGTTGGAAGATC-39 and NotI 59-TGGAAGAATTCGCGGCCGCAG-39. A final fragment of 2366 bp (SHR5 39) was amplified
and cloned into the pGEM-T vector (Promega) and its sequence was
determined using automated sequencing. Searches for the full-length
cDNA sequence of SHR5 were performed at the S. officinarum
database of TIGR Gene Indices using BLASTN (Altschul et al.,
1997). The clone CA116269 (GenBank accession no.) was identified
as SHR5, and its 59 cDNA sequence was used to construct the entire
SHR5 cDNA (GenBank accession no. DQ067098). A search for
SHR5-related sequences was performed at the NCBI World Wide
Web server using BLASTX in the non-redundant database. The best
hits were used for further analysis. Derived protein sequences were
analysed using BLAST2seq (Tatusova and Madden, 1999) to
determine the percentage of similarity between the sequences.
Pfam (Bateman et al., 2004) and SMART (Letunic et al., 2004)
were used to identify protein domain architectures. Hydropathy
analysis was generated by the Kyte–Doolittle algorithm (Kyte and
Doolittle, 1982). Multiple sequence alignments were carried out
using CLUSTAL3(Thompson et al., 1997). Phylogenetic analyses
were conducted and viewed using MEGA version 2.1 (Kumar et al.,
2001) based on the Neighbor–Joining method.
Semi-quantitative RT-PCR
Ten ll of the first-strand cDNA reaction diluted four times was used
in a standard 50 ll PCR reaction (10 mM TRIS-Cl pH 8.4, 50 mM
KCl, 1.5 mM MgCl2, 0.01% gelatin, and 2.5 U Taq polymerase) with
200 ng of the specific primers. The primers used were: SHR5.fwr
(59-TGACCGAGCACTCTTTGGTAA-39) and the 39-UTR based
SHR5.rev (59-TCGAATTAATCCAGCAGCAGC-39), or ubi1 (59ATGCAGATCTTTGTGAAGAC-39) and ubi2 (59-TTACTGACCACCACGAAGAC-39). In order to carry out experiments on the
exponential phase of RT-PCR reaction curves, standard curves
were performed varying the amount of cDNA and the number of
cycles of PCR reaction. PCR conditions were 94 8C for 5 min,
followed by 25–35 cycles (94 8C for 1 min, 55 8C for 1 min, 72 8C for
1 min) and with 72 8C for 5 min. The polyubiquitin constitutive gene
was used as an internal control in PCR reactions. Products of the PCR
reactions were eletrophoretically separated on 1% agarose gel,
visualized with ethidium bromide under UV light, and then transferred onto a nylon membrane and hybridized with a radioactivelylabelled cDNA fragment from sugarcane SHR5 or a ubiquitin cDNA
fragment from Arabidopsis thaliana. Densitometric analyses of
bands obtained in PCR amplification were performed using Scion
Image 4.0.2 software (Copyrightª 2000 Scion Corporation).
561
GraphPad Prism (GraphPad Software Inc, San Diego, CA, USA)
was used to perform statistical analysis. When three or more groups
were analysed, samples were compared using one-way analysis of
variance (ANOVA) followed by Tukey test; when two groups were
compared, the unpaired t-test was used. A P value <0.05 was
considered significant.
Results
Isolation and sequence analysis of SHR5 cDNA
cDNA-AFLP experiments were carried out in order to
detect sugarcane genes that were repressed and/or induced
in association with endophytic bacteria. RNA from four
groups of sugarcane plantlets inoculated or not inoculated
with the following different species of endophytic bacteria:
H. rubrisubalbicans (HR), H. seropedicae (HS+) and
H. seropedicae mutant, which is unable to fix N2 (HS–),
were compared. Twelve transcript-derived fragments
(TDF) were isolated and sequenced (F Vinagre, unpublished results). A 179bp TDF, corresponding to SHR5,
showed homology with LRR domains and was selected to
be characterized further.
As shown in Fig. 1A, the SHR5 cDNA band is only
detected in non-inoculated plants (CON), suggesting that
SHR5 gene expression is being repressed in plants inoculated with any one of the endophytic bacteria mentioned
above. As the SHR5 TDF corresponds to a small region of
the SHR5 complete cDNA, based on its DNA sequence
information an attempt was made to obtain a longer SHR5
cDNA sequence using two different approaches. A 2366 bp
long cDNA corresponding to the 39 end of SHR5 complete
cDNA was obtained by 39-Nested PCR. In addition, an EST
corresponding to SHR5 was identified in the database of
TIGR Gene Indices (GenBank accession no. CA116269)
and its 59 cDNA sequence was used to construct the SHR5
cDNA sequence (GenBank accession no. DQ067098).
Although the first methionine was not sequenced in EST
CA116269, it is very likely that only a few amino acids
were missing, because the signal peptide was identified in
the coding region and a comparison with other homologue
proteins revealed a protein of similar size. The SHR5 cDNA
has an estimated open reading frame of approximately 3084
bp, coding for a predicted protein of 1027 amino acids and
a deduced molecular mass of approximately 107 kDa
including the signal peptide (Fig. 1B). Analyses of
structural properties of the SHR5 predicted protein using
Pfam (Bateman et al., 2004) and SMART programs
(Letunic et al., 2004) suggest that SHR5 encodes an RLK
protein with four distinct regions: an N-terminal hydrophobic signal peptide, extracellular leucine-rich repeats (LRR),
a transmembrane domain (TM), and a cytoplasmatic kinase
domain (Fig. 1B, C). In the extracellular region, the first
hydrophobic domain (black box) corresponds to the signal
peptide (SP) possibly responsible for targeting the protein
into the endoplasmic reticulum (Von Heijne, 1991). The
562 Vinagre et al.
Fig. 1. SHR5 identification and sequence analysis. (A) Visualization of a part of a cDNA-AFLP autoradiogram showing the TDF corresponding to
SHR5 cDNA (asterisk). Four lanes are shown, corresponding to an amplified template from sugarcane plantlets of the B-4362 variety inoculated with
endophytic bacteria. HR: H. rubrisubalbicans; HS+: H. seropedicae; HS–: H. seropedicae mutant unable to fix N2; CON: plants free of micro-organisms.
(B) Predicted amino acid sequence of SHR5. Non-polar regions corresponding to the signal peptide sequence and transmembrane region are denoted by
black boxes. Italicized amino acids indicate LRRs. The sequence corresponding to the TDF cDNA is dash underlined. Kinase domain is highlighted with
grey. Roman numerals indicate the 11 characteristic subdomains of the protein kinases (Hanks et al., 1988). Amino acids highly conserved among protein
kinases are indicated by asterisks. Residues that indicate serine/threonine specificity are underlined. (C) Structural features of the SHR5 protein. A
hydropathy plot was deduced from the SHR5 amino acid sequence, where increased hydrophobicity is denoted by positive values. A linear schematic
representation of SHR5 denotes the signal peptide (SP), LRRs, transmembrane (TM), and kinase domains identified by computational analysis.
SHR5 extracellular region contains eight predicted LRR
domains. LRR domains can have a role in ligand recognition, protein–protein interactions and agonist binding,
in proteins involved in signal transduction in eukaryotes
(Baker et al., 1997). The second hydrophobic region,
encompassing a segment of 23 amino acids, is located in
the middle of the predicted protein sequence, corresponding
to a single-membrane spanning region. Figure 1C represents
RLK in plant–endophytic bacteria association
a hydropathy plot generated by the Kyte–Doolittle algorithm
(Kyte and Doolittle, 1982), where increased hydrophobicity
is denoted by positive values showing the hydrophobic
regions corresponding to the signal peptide and transmembrane domain. The kinase catalytic domain (from amino acid
694 to 962) was detected in the intracellular region of the
protein. It is well known that, in their catalytic domain,
serine/threonine/tyrosine protein kinases display amino acid
sequence similarities that consist of 11 conserved subdomains (Hanks et al., 1988). In the predicted SHR5 protein,
these 11 potential kinase subdomains are present (identified
by roman numerals in Fig. 1B). Furthermore, the amino acid
sequences in subdomains VIb (DIKASN) and VIII
(GTFGYLAPE) are consistent with the consensus sequences, DLKXXN and G(T/S)XX(Y/F)XAPE, respectively,
which are most common among serine/threonine kinases.
This result suggests that SHR5 may have serine/threonine
rather than tyrosine substrate specificity.
Searches at the NCBI database were performed in order
to identify protein sequences closely related to the extracellular domain, the kinase domain, and the entire SHR5
predicted protein. These three different analytical approaches revealed that the closer relatives to SHR5 are
putative LRR-RLKs from rice (O. sativa) and A. thaliana,
the kinase domain being the region with the most conserved
homology. The closest homologue of SHR5 is a rice protein
(GenBank accession no. XP_480586) that has 1030 amino
acids and exhibits 77% identity and 86% similarity with
SHR5. In A. thaliana, the closest SHR5 homologue
(GenBank accession no. NP_176009) has 1032 amino acids
and shows 53% identity and 68% similarity with SHR5.
To classify SHR5 within the 15 LRR-RLK subfamilies
established by Shiu et al. (2004), a phylogenetic tree using
the kinase domain sequences of representative Arabidopsis
members of each LRR-RLK subfamily was generated
according to Shiu and Bleecker (2001b). With a bootstrap
value of 100%, SHR5 formed a well-supported clade with
LRR protein of the VIII-2 subfamily, indicating that SHR5
belongs to this LRR subfamily (data not shown). To obtain
ideas about the possible biological roles of SHR5, another
phylogenetic tree was generated based on kinase domains
of the previously described plant LRR-RLK proteins
involved in development, symbiosis, and host defence,
as revised by Diévart and Clark (2004). In this analysis,
other sugarcane LRR-RLK-related proteins were included
(Fig. 2). Sugarcane SHR5, together with its homologues in
rice and A. thaliana, formed a well-supported branch,
separated from the other genes with known biological roles,
with a bootstrap value of 100% (detailed with a box in
Fig. 2). These data suggest that the SHR5 gene encodes for
a novel LRR-RLK protein not yet associated with any
biological role described for LRR-RLK proteins to date.
The other sugarcane proteins were grouped with different
classes of LRR-RLK, showing that sugarcane has a diverse
collection of genes encoding for LRR receptor-like kinases.
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94
86
100
100
100
100
98
100
81
98
100
98
99
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PSK Dcar
TC54254 Sc
EXS Ath
VH1 Ath
TC59412 Sc
BRI1 Ath
SR160 Lesc
INRPK1 Inil
ERECTA Ath
TC49209 Sc
BAK1 Ath
SERK1 Ath
TC49873 Sc
SHR5 Ath
SHR5 Sc
SHR5 Osat
HAESA Ath
TC58950 Sc
HAR1 Ljap
CLV1 Ath
TC65555 Sc
NORK Msat
SYMRK Ljap
FLS2 Ath
Xa21 Osat
TC70496 Sc
Fig. 2. Phylogenetic analyses of LRR-RLKs. Neighbor–Joining tree,
including many LRR-RLKs of known function, sugarcane LRR-RLKrelated sequences, sugarcane SHR5 and its homologues in A. thaliana
and rice, was created using the MEGA program. To evaluate the
confidence limits of the internal branches of the tree, a bootstrap analysis
with 2000 replications was performed on the data set. The numbers next
to the nodes give bootstrap percentages. This is a condensed tree with
a cutoff value of 80. Accession numbers: BAK1_Ath (Q94F62);
BRI1_Ath (AAC49810); CLV1_Ath (AAB58929); ERECTA_Ath
(AAC49302); EXS_Ath (Q9LYN8); FLS2_Ath (AB010698);
HAESA_Ath (AAA32859); HAR1_Ljap (BAC41331); INRPK1_Inil
(AAB36558); NORK_Msat (CAD10807) PSK_Dcar (BAC00995);
SERK1_Ath (CAB42254); SHR5_Ath (NP_176009); SHR5_Osat
(BAD02994); SHR5_Sc (DQ067098); SR160_Lesc (Q8GUQ5);
SYMRK_Ljap (AAM67418); VH1_Ath (Q9ZPS9); Xa21_Osat
(A57676). Sugarcane (Saccharum spp) RLKs related sequences are
shown with their corresponding TC identifiers (TIGR Gene Indices). Ath,
A. thaliana; Dcar, Daucus carota; Inil, Ipomoea nil; Lesc, Lycopersicon
esculentun; Ljap, Lotus japonicus; Msat, Medicago sativa; Osat, Oryza
sativa; Sc, Saccharum spp.
SHR5 gene expression during association between
sugarcane and endophytic nitrogen-fixing bacteria
In order to determine how SHR5 mRNA expression is
modulated during plant colonization by endophytic diazotrophic bacteria, sugarcane in vitro-grown plantlets of the
SP70-1143 variety, free of micro-organisms, were inoculated with different species of endophytic diazotrophic
bacteria. SP70-1143 is a sugarcane variety described to
show high levels of BNF contribution (Urquiaga et al.,
1992). SHR5 mRNA levels were investigated in these
samples by semi-quantitative RT-PCR (Fig. 3). SHR5
mRNA levels decreased (0.26560.035) in sugarcane plants
associated with G. diazotrophicus PAL5 (GD), compared
with mRNA levels in non-inoculated plants. Inoculation
with Herbaspirillum spp., H. rubrisubalbicans HCC103
strain (HR), and H. seropedicae HRC54 strain (HS),
showed an even more significant decrease in SHR5
mRNA levels, greater than 90% (0.07560.075), when
564 Vinagre et al.
compared to the control. As field plants are naturally
colonized by a mixture of different species of bacteria,
sugarcane plants were also inoculated concomitantly with
the three bacterial species mentioned above (GR; HR; HS).
SHR5 mRNA levels decreased 95% in the inoculated
plantlets (0.0560.05) (Fig. 3). The analysis was also performed with in vitro-grown sugarcane plantlets inoculated
with the A. brasilensis Sp245 strain (AZO), a nitrogenfixing Azospirillum strain that can also be endophytic
(James and Olivares, 1998). The inoculation with AZO
led to a 69% decrease of SHR5 expression (0.3160.06). In
all the experiments, plant colonization by the inoculated
bacteria was confirmed by the Most Probable Number
(MPN) estimation (Materials and methods). Rates of
colonization always ranged between log 4–6 cfu gÿ1 fwt
(data not shown).
The data show that SHR5 is widely repressed in plants
colonized by the endophytic diazotrophic bacteria tested. In
addition, the decreased mRNA expression is not specific to
a bacterial species.
SHR5 gene expression in response to auxins
It is well known that endophytic nitrogen-fixing bacteria are
capable of producing auxin (Fuentes-Ramirez et al., 1993)
and that this can be one of the mechanisms by which these
bacteria promote plant growth. Furthermore, it is known
that RLKs are involved in hormone signalling in plants
(Matsubayashi et al., 2002; Li et al., 2002). To verify if the
auxin produced by these bacteria is directly modulating
SHR5 gene expression, semi-quantitative RT-PCR were
performed on plantlets grown for 7 d with two different
auxins in the medium: indole acetic acid (IAA) and
naphthalene acetic acid (NAA), both at concentrations
Fig. 3. SHR5 gene expression during association between sugarcane
and endophytic nitrogen-fixing bacteria. Sugarcane plantlets of the
SP70-1143 variety were inoculated with: GD, G. diazotrophicus;
HR/HS, Herbaspirillum spp; MIX, mixture of GD, HR, and HS; AZO,
A. brasilensis; CONT, plants free of micro-organisms. Ubiquitin was
used as an internal control. The graph represents the average of two experiments with SHR5 mRNA levels corrected for ubiquitin mRNA levels,
comparing all groups to control levels. Error bars indicate 6standard
error. Different letters indicate significant differences between groups.
similar to those secreted by bacteria in culture medium
(Fuentes-Ramirez et al., 1993) (Fig. 4). SHR5 mRNA
expression was not modified in either one of the treatments
when compared with control mRNA expression (IAA:
1.11560.065; NAA: 0.9860.12). These results suggest
that the mechanism responsible for modulation of SHR5
expression does not involve hormone produced by bacteria.
SHR5 gene expression in response to association with
endophytic nitrogen-fixing bacteria in different
sugarcane genotypes
To evaluate if the efficiency of the association between
sugarcane and the endophytic diazotrophic bacteria would
affect SHR5 expression, sugarcane genotypes with different
levels of BNF contribution were investigated. SHR5 mRNA
levels were compared by semi-quantitative RT-PCR experiments in in vitro-grown plantlets of varieties with high BNF,
SP70-1143 (SP), and low BNF, Chunee (CH), inoculated with
the endophytic nitrogen-fixing bacteria G. diazotrophicus
(GD) (Fig. 5). Interestingly, there was no difference in total
bacteria colonization counts between the two different
genotypes (data not shown). SHR5 mRNA expression was
repressed in both genotypes (Fig. 5A). Nevertheless, it
strikingly decreased in the SP70-1143 variety (0.0860.02)
compared with Chunee plantlets (0.73560.005) (Fig. 5A).
This result indicates that the two genotypes, which have
different levels of BNF contribution, do not respond in the
same way to the association with endophytic diazotrophic
Fig. 4. SHR5 gene expression in response to auxins. Sugarcane plantlets free of micro-organisms were cultivated in inoculation medium
supplemented with 2,4 lg mlÿ1 of auxin (IAA or NAA) for 7 d. IAA,
indole acetic acid; NAA, naphthalene acetic acid; CONT, plants
cultivated in medium without hormones. Ubiquitin was used as an
internal control. The graphs represent the average of two experiments with SHR5 mRNA levels corrected for ubiquitin mRNA levels.
Error bars indicate 6standard error. Different letters indicate significant
differences between groups.
RLK in plant–endophytic bacteria association
bacteria. Remarkably, SHR5 mRNA levels in SP70-1143
control plantlets are 81% lower (0.19560.025) than in
Chunee control plantlets, suggesting that the expression of
SHR5 in SP70-1143 is normally kept lower than in the
Chunee genotype. SHR5 expression was also investigated in
a pathogenic interaction with a diazotrophic endophyte. The
sugarcane B-4362 variety is known to develop the mottled
stripe disease when associated with H. rubrisubalbicans
(HR) (Olivares et al., 1997). This susceptibility is exclusive
to this bacterial species, and is not triggered by other species
of the Herbaspirillum genus. SHR5 mRNA levels were
measured by semi quantitative RT-PCR in B-4362 in vitrogrown plantlets inoculated with two different Herbaspirillum species: H. rubrisubalbicans (HR) and H. seropedicae
(HS) (Fig. 5B). The results showed that SHR5 expression
was significantly diminished in healthy B-4362 plantlets
colonized by HS (0.2960.07) when compared with the
control, while colonization by HR that led to development of
disease revealed a non-significant decrease of SHR5 mRNA
expression (0.6560.08). Taken together, the data suggest
that SHR5 mRNA levels in plant cells are related to the
efficiency of the association between sugarcane and endophytic bacteria, meaning that the more beneficial the
association is for the plant, the more pronounced the
decrease is in SHR5 expression.
SHR5 expression in response to different
micro-organisms and pathogenic interactions
565
bacteria as opposed to with any other micro-organism
interaction, SHR5 mRNA levels were investigated in sugarcane plantlets inoculated with different micro-organisms.
Agrobacterium tumefaciens A281 strain (AGR) is a
nitrogen-fixing bacterium, pathogenic in dicots, which
does not cause disease in sugarcane (Kanvinde and Sastry,
1990). Plantlets inoculated with AGR (0.9260.04) showed
no changes on SHR5 mRNA levels compared with control
non-inoculated plants (Fig. 6A). Leifsonia xyli subsp.
Xyli (LEIF) is a pathogenic bacterium responsible for the
ratoon-stunting sugarcane disease, and exhibits colonization properties similar those seen in endophytic bacteria
(Harrison and Davis, 1986). So far, L. xyli subsp. Xyli has
only been identified in association with sugarcane and does
not appear to be a soil-borne pathogen (Gillaspie and
Teakle, 1989). Colonization of SP70-1143 with LEIF
showed the same pattern of SHR5 expression (1.0460.06)
observed in plants free of micro-organisms (Fig. 6B).
Sugarcane plants colonized by Xanthomonas albilineans,
the pathogenic bacteria responsible for the leaf scald disease
(Ricaud and Ryan, 1989), exhibited a small, but not
significant increase of SHR5 expression (1.3760.17),
compared with control SHR5 mRNA levels (Fig. 6C). The
modulation of SHR5 expression during sugarcane interaction with micro-organisms other than bacteria on SHR5
expression was also investigated. Inoculation of sugarcane
plantlets of SP71-799 variety with Puccinia melanocephala
(PUC) fungi, the causal agent of sugarcane rust disease
To verify if the modulation of SHR5 gene expression was
associated only with beneficial endophytic diazotrophic
Fig. 5. SHR5 gene expression in response to association with endophytic nitrogen-fixing bacteria in different sugarcane genotypes. (A)
Sugarcane plantlets of the SP70-1143 variety (high BNF) and of Chunee
specie (low BNF) inoculated with endophytic bacteria. (B) Sugarcane
plantlets of the B-4362 variety (susceptible to H. rubrisubalbicans) were
inoculated with endophytic bacteria. CH, Chunee; SP-, SP70-1143;
GD, G. diazotrophicus; HR, H. rubrisubalbicans; HS, H. seropedicae;
CONT, plants free of micro-organisms. Ubiquitin was used as an internal
control. The graphs represent the average of two experiments with SHR5
mRNA levels corrected for ubiquitin mRNA levels, comparing all groups
to control levels. Error bars indicate 6standard error. Different letters
indicate significant differences between groups.
Fig. 6. SHR5 expression in response to different micro-organisms and
pathogenic interactions. (A) Sugarcane plantlets of SP70-1143 variety
inoculated with: AGR, A. tumefaciens; CONT, plants free of microorganisms. (B) Sugarcane plantlets of SP70-3370 variety inoculated with:
LEIF, Leifsonia xyli subsp. Xyli; CONT, plants free of micro-organisms. (C)
Sugarcane plants of SP80–1842 variety colonized by: XANT, Xanthomonas albilineans; CONT, plants free of X. albilineans. (D) Sugarcane
plantlets of SP71-799 variety inoculated with: PUC, Puccinia melanocephala; CONT, plants free of micro-organisms. (E) Sugarcane plants of
SP86-155 variety colonized by: MOS, sugarcane mosaic virus; CONT,
plants free of mosaic virus. Ubiquitin was used as an internal control. The
graphs represent the average of two experiments with SHR5 mRNA levels
corrected for ubiquitin mRNA levels. Error bars indicate 6standard error.
Different letters indicate significant differences between groups.
566 Vinagre et al.
(Purdy et al., 1983), exhibited a slight, but not significant,
increase of SHR5 expression (1.4160.11), compared with
control SHR5 mRNA levels (Fig. 6D). Shoots from plants
with the Sugarcane Mosaic Virus, SCMV (MOS) responsible for the Sugarcane Mosaic Virus Disease (Comstock
and Lentini, 2002) showed no significant difference in
SHR5 mRNA expression (0.99560.155) in relation to shoots
of virus-free plants (Fig. 6E). Taken together, these results
indicate that SHR5 expression is modulated only in interactions between sugarcane and beneficial endophytic bacteria.
SHR5 expression in abiotic stress
In order to investigate if SHR5 gene expression responds to
abiotic stress, semi-quantitative RT-PCR analyses of SP701143 in vitro-grown sugarcane plantlets submitted to saline
and temperature stresses were carried out (Fig. 7). For 16 h,
neither the 1% NaCl nor the 2% NaCl saline treatments
affected SHR5 expression significantly, in relation to nontreated plants (1.02560.135 and 128060.05, respectively)
(Fig. 7A). Plants kept at 50 8C for 15 min did not show any
significant difference in SHR5 mRNA levels in relation to
control (1.0560.08). Plants incubated at 45 8C for 15 min
showed an increase of 38% (1.37560.075) in SHR5
expression when compared with plants kept in normal
conditions (Fig. 7B). This modulation on SHR5 expression
differs from the one observed with endophytic bacteria
association, which leads to a decrease on gene expression.
Discussion
In this study, the isolation of SHR5, a novel cDNA which
encodes a putative, receptor-like protein kinase (RLK) from
sugarcane is reported. The predicted SHR5 structural fea-
Fig. 7. SHR5 expression in abiotic stress. Sugarcane plantlets free of
micro-organisms were submitted to: (A) Heat shock treatment at 45 8C or
50 8C for 15 min; (B) saline stress treatment at 1% or 2% NaCl for 16 h.
CONT, plants were maintained at normal conditions. Ubiquitin was used
as an internal control. The graphs represent the average of two
experiments with SHR5 mRNA levels corrected for ubiquitin mRNA
levels. Error bars indicate 6standard error. Different letters indicate
significant differences between groups.
tures, including a hydrophobic signal peptide, an extracellular domain, a hydrophobic membrane-spanning segment
and a highly conserved kinase domain, strongly suggest that
the encoded protein is a receptor protein kinase (Fig. 1).
Computer analysis of the kinase domain revealed, among
others, two subdomains: VIb (DIKASN) and VIII (GTFGYLAPE), suggesting that SHR5 has serine/threonine kinase
substrate specificity. Most RLK genes described in plants, so
far, encode for serine/threonine kinases (Shiu and Bleecker,
2001b). The SHR5 putative extracellular domain contains
eight leucine-rich repeats (LRR), placing this receptor into
the LRR-RLK family. Phylogenetic analysis of the SHR5
kinase domain classified it as belonging to the LRR VIII-2
subfamily (data not shown) (Shiu et al., 2004). So far, no
studies have been reported associating any biological
functions with the members of this subfamily. Even though
the phylogeny used to classify RLKs into subfamilies was
based on the kinase domain, it showed that members within
each of the RLK subfamilies tended to have similar
extracellular domains, such as the structural arrangement
of LRRs (Shiu and Bleecker, 2001b). LRR motifs are
thought to be involved in protein–protein interactions, and
all its ligands described so far in plants are proteins, except
for a plant steroid hormone (brassinolide) (Matsubayashi,
2003), although evidence suggests the involvement of
a putative secreted brassinosteroid-binding protein in the
binding of this hormone to its LRR receptor (Li et al., 2001).
So far, LRR-RLK proteins have few reported ligands, and
none of them belong to the LRR-VII subfamily. According
to Pfam, SHR5 has unrelated sequences or ‘islands’ between
their LRR motifs; the first ‘island’ being between the first and
second LRR motifs. The second island is between the fourth
and fifth LRRs. The BRI1 receptor also presents an LRR
island that might mediate the binding of this receptor to its
steroid hormone ligand, brassinosteroid (Wang et al., 2001).
These data suggest that SHR5 LRR islands could also be
related to ligand-binding. It is interesting to note that, in the
phylogenetic analysis (Fig. 2), SHR5 was grouped with its
homologues in other species, like the dicot A. thaliana, rather
than with other sugarcane putative RLKs or other RLKs of
known functions. This indicates that SHR5 might represent
a new class of gene present in a wide range of different
species. An example of conservation has already been
reported in the LRR-RLK function between monocots and
dicots (Yamamuro et al., 2000).
These data suggest that this new RLK might play a role in
the association of plants with beneficial endophytic bacteria.
It has been shown that the sugarcane variety SP70-1143, in
association with the beneficial endophytic diazotrophic
bacteria G. diazotrophicus (GD), Herbaspirillum spp (HR/
HS), and Azospirillum brasilensis (AZO) (Fig. 3), exhibited
significantly decreased levels of SHR5 mRNA. Sugarcane in
the field is colonized by multiple species of endophytic
bacteria (Baldani et al., 1997), and these results showed that
inoculation with a mixture of different species of bacteria led
RLK in plant–endophytic bacteria association
to similar expression levels seen with individual species.
The response of SHR5 expression to the association with the
beneficial endophytic bacteria seems to be dependent on the
plant genotype. SP70-1143 and Chunee sugarcane genotypes, described as having high and low biological nitrogen
fixation (BNF) rates, respectively (Urquiaga et al., 1992),
showed different modulation of SHR5 gene expression (Fig.
5A). When inoculated with GD, both genotypes showed
a significant decrease in mRNA expression, but this decrease was considerably more pronounced in the SP70-1143
genotype. Comparing mRNA levels in plants free of
endophytes (controls), it was observed that SHR5 is much
less expressed (81%) in SP70-1143 than in Chunee, indicating that SHR5 steady-state mRNA levels are lower in
plants with high BNF rates. Chunee is a wild species not
used commercially and SP70-1143, which has an important
commercial value in Brazil, is a sugarcane variety selected
for its high yields. Historically, Brazilian sugarcane cultivars
have been selected for their greater yield with low inputs of
inorganic nitrogen fertilizer (Cavalcante and Döbereiner,
1988), which is possibly achieved by choosing genotypes
that establish more efficient associations with diazotrophic
micro-organisms. It is possible that a decreased expression
of SHR5 in SP70-1143 is a consequence of this selection, as
it is correlated with a more successful plant–endophytic
bacteria interaction.
An intriguing question is how plants sense a beneficial
micro-organism. Metabolites provided by the endophytes
could be candidates to take part in this signalling. Incubation of sugarcane plantlets with two different auxins
(IAA and NAA) in the medium caused no difference in
SHR5 expression when compared with non-treated plantlets
(Fig. 4). In addition, cDNA AFLP analyses showed that
plants inoculated with a mutant H. seropedicae bacterium
unable to fix N2 also exhibited decreased SHR5 gene
expression (Fig. 1A). These data suggest that the SHR5
response to the association with beneficial endophytes may
not be directly attributed to nitrogen fixation or auxin
production by the bacteria, but possibly to signalling
mechanisms recognized during the interaction with the
beneficial micro-organism. Interestingly, it seems that the
decrease in SHR5 expression is not only dependent on
the interaction between the plant and potentially beneficial bacteria, but also that it is mainly controlled by the effective establishment of an efficient association. Inoculation
of B-4362 with H. rubrisubalbicans (HR), which causes
mottled stripe disease in this plant genotype, led to an insignificant decrease in SHR5 mRNA expression; meanwhile,
inoculation of B-4362 with H. seropedicae (HS), which establishes a non-pathogenic interaction, caused a considerable
decrease in SHR5 mRNA levels.
The modulation of SHR5 gene expression does not seem
to be a general response to stress, since it does not respond
to the abiotic stress imposed by saline and heat treatments.
Only plants exposed to 45 8C showed increased SHR5
567
mRNA expression. This increase is possibly triggered by a
different response pathway, since the association with
endophytic bacteria decreases SHR5 mRNA levels. An
LRR-RLK gene from Arabidopsis has been reported to be
induced by abiotic stresses such as dehydration, high salt,
and cold treatments (Hong et al., 1997), indicating that
LRR-RLKs may be involved in multiple-stress signal transduction. In addition, the studies on modulation of SHR5
expression by interaction with other micro-organisms revealed that associations with non-pathogenic diazotrophic
bacteria (A. tumefaciens), pathogenic endophytic bacteria (L.
xyli subsp. Xyli and X. albilineans), pathogenic fungus (P.
melanocephala) or a pathogenic virus (SCMV) did not alter
SHR5 mRNA expression (Fig. 6), which strengthens the idea
that SHR5 expression is only decreased in beneficial
associations between sugarcane and endophytic bacteria.
It is interesting to point out that receptors related to host
defence or symbiosis are structurally similar. Therefore, the
possibility cannot be excluded that SHR5 might be involved in a plant defence mechanism and that the decrease
in SHR5 gene expression may be a necessary process for
the establishment of an efficient association between
sugarcane and endophytic bacteria. It has been hypothesized that during the first interaction between plant and
symbiotic bacteria, the latter is recognized as a potential
pathogen and host defence mechanisms are activated. Later,
when a beneficial association is established, those mechanisms are suppressed (Sikorski et al., 1999).
It is important to highlight that, only recently, genes
encoding LRR-RLKs have been reported to be related to
beneficial interactions between plant and micro-organisms.
For example, NORK/SYMRK leguminous mutants are
unable to establish symbiosis with arbuscular mycorrhizal
(AM) fungi or rhizobia (Endre et al., 2002; Stracke et al.,
2002). AM-like interactions were detected in early land
plants whereas root nodule symbioses evolved later
(Kistner and Parniske, 2002). Proteins that possess similarity with the NORK/SYMRK extracellular domain are
found in Arabidopsis, monocots, and gymnosperms (Endre
et al., 2002), suggesting that, during evolution, an ancient
system of signal transduction was recruited for symbiosis
establishment. Resembling NORK/SYMRK, SHR5 is also
an LRR-RLK and orthologues are possibly found in
monocots and Arabidopsis (Fig. 2). Some symbiosisrelated RLKs, despite structural differences from SHR5,
can have their mRNA levels decreased in plants colonized
by their symbionts, as occurs with SHR5 gene expression
(Lange et al., 1999; Radutoiu et al., 2003). Therefore, the
data suggest that SHR5 may take part in a novel signalling
cascade involved in the establishment of symbiotic-like
interactions between plants and micro-organisms.
Despite the fact that modulation of SHR5 expression has
been primarily by micro-organisms, it can be expected that
SHR5 may also be involved in development processes, as
a great number of the LRR-RLK genes described so far are
568 Vinagre et al.
associated to development. Overlaps between signalling
pathways can occur among development and host defence
signalling proteins (Gomez-Gomez et al., 1999; Montoya
et al., 2002; Pastuglia et al., 2002). Moreover, HAR1/
NARK LRR-RLK is thought to be involved in the regulation of the later stages of nodulation and also in root
development, as HAR1/NARK non-inoculated mutants
have a shortened root system and an enhanced number of
lateral roots (Krusell et al., 2002; Nishimura et al., 2002;
Searle et al., 2003; Wopereis et al., 2000).
Further studies are required in order to understand the
mechanisms that involve the SHR5 signalling pathways.
SHR5 could be involved directly in plant–bacteria signalling, recognizing molecules produced by bacteria or recognizing bacteria themselves, so it is also important to
identify the SHR5 ligands. On the other hand, SHR5 could
be involved downstream of a primary bacteria recognition
event with its down-regulation being an intermediate step in
the signalling pathway that leads to a successful plant–
bacteria interaction. Future analysis of intracellular target
molecules interacting with SHR5 could help elucidate these
signalling mechanisms.
In this work, it has been reported that SHR5 encodes an
LRR-RLK that seems to be related to the mediation of the
beneficial association between plant and endophytic bacteria. Besides contributing to the understanding of mechanisms underlying endophytic association, this work may
also provide tools for future agricultural applications.
Genetic handling of genes that can lead to more efficient
plant–bacteria associations could result in higher production yields, reduced input costs, and diminished negative
environmental consequences due to the use of the excessive
addition of N fertilizers.
Acknowledgements
We are grateful to Leonardo Mega Francxa and Ana Cláudia de Jesus
for technical assistance in DNA sequencing and plant culture,
respectively. We also thank COPERSUCAR for providing sugarcane
genotypes and plant material infected with P. melanocephala and
mosaic virus disease and to Goncxalo Apolinário da Silva for
providing plant material infected with L. xyli subsp. Xyli. We thank
Ana Carolina Andrade for help in finalizing the manuscript and
Erika M Korowin (USA) for language editing. FV is indebted to the
Conselho Nacional de Desenvolvimento Cientı́fico e Tecnológico
(CNPq) for a graduate fellowship. ASH and PCGF received support
from a CNPq research grant. The study was partially supported by
the project PronexII/CNPq and PADCT III.
References
Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z,
Miller W, Lipman DJ. 1997. Gapped BLAST and PSI-BLAST:
a new generation of protein database search programs. Nucleic
Acids Research 25, 3389–3402.
Bachem CW, van der Hoeven RS, de Bruijn SM, Vreugdenhil D,
Zabeau M, Visser RG. 1996. Visualization of differential gene
expression using a novel method of RNA fingerprinting based on
AFLP: Analysis of gene expression during potato tuber development. The Plant Journal 9, 745–753.
Baker B, Zambryski P, Staskawicz B, Dinesh-Cumar SP. 1997.
Signalling in plant microbe interactions. Science 276, 726–733.
Baldani JI, Caruso L, Baldani VLD, Goi SR, Döbereiner J. 1997.
Recent advances in BNF with non-legume plants. Soil Biology and
Biochemistry 29, 911–922.
Baldani JI, Pot P, Kirchlaof G, et al. 1996. Emended description of
Herbaspirillum; inclusion of [Pseudomonas] rubrisubalbicans,
a mild plant pathogen as Herbaspirillum rubrisubalbicans comb.
Nov. and classification of a group of clinical isolation (EF group 1)
as Herbaspirillum species 3. International Journal of Systems
Evolution Microbiology 46, 802–810.
Baldani VLD, Baldani JI, Olivares FL, Döbereiner J. 1992.
Identification and ecology of Herbaspirillum seropedicae and the
closely related Pseudomonas rubrisubalbicans. Symbiosis 13, 65–73.
Bateman A, Coin L, Durbin R, et al. 2004. The Pfam protein families
database. Nucleic Acids Research Database Issue 32, D138–D141.
Boddey RM, Döbereiner J. 1995. Nitrogen fixation associated with
grasses and cereals: recent progress and perspectives for the future.
Fertilizer Research 42, 241–250.
Cavalcante VA, Döbereiner J. 1988. A new acid-tolerant nitrogenfixing bacterium associated with sugarcane. Plant and Soil 108,
23–31.
Comstock JC, Lentini RS. 2002. Sugarcane mosaic virus disease.
Agronomy Department, Florida Cooperative Extension Service,
Institute of Food and Agricultural Sciences, University of Florida.
SS-AGR-209.
Diévart A, Clark SE. 2004. LRR-containing receptors regulating
plant development and defence. Development 131, 251–261.
Endre G, Kereszt A, Devei Z, Mihacea S, Kalo P, Kiss G. 2002. A
receptor kinase gene regulating symbiotic nodule development.
Nature 417, 962–966.
Fuentes-Ramirez LE, Jimenez-Salgado T, Abarca-Ocampo IR,
Caballero-Mellado J. 1993. Acetobacter diazotrophicus, an
indolacetic acid-producing bacterium isolated from sugar cane
cultivars in Mexico. Plant and Soil 154, 145–150.
Gillaspie AG, Teakle DS. 1989. Ratoon stunting disease. In: Ricaud
C, Egan BT, Gillaspie Jr AG, Hughes CG, eds. Diseases of
sugarcane: major diseases. New York: Elsevier Science Publishing Inc., 59–80.
Gomez-Gomez L, Felix G. Boller T. 1999. A single locus
determines sensitivity to bacterial flagellin in Arabidopsis thaliana. The Plant Journal 18, 277–284.
Hanks SK, Quinn AM, Hunter T. 1988. The protein kinase family
conserved features and deduced phylogeny of the catalytic domain.
Science 241, 42–52.
Harrison NA, Davis MJ. 1986. Patterns of colonization of vascular
tissues by clavibacter-xyli subsp. xyli in stalks of sugarcane
cultivars differing in susceptibility to ratoon stunting disease.
Phytopathology 76, 1136–1136.
Hendre KR, Iyer RS, Kotwain M, Kluspe SS, Mascarenhas AF.
1983. Rapid multiplication of sugarcane by tissue culture.
Sugarcane 1, 5–8.
Hong SW, Jon JH, Kwak JM, Nam HG. 1997. Identification of
a receptor-like protein kinase gene rapidly induced by abscisic
acid, dehydration, high salt, and cold treatments in Arabidopsis
thaliana. Plant Physiology 113, 1203–1212.
James EK, Olivares FL. 1998. Infection and colonization of sugar
cane and other graminaceous plants by endophytic diazotrophs.
Critical Reviews in Plant Sciences 17, 77–119.
James EK, Reis VM, Olivares FL, Baldani JI, Dobereiner J.
1994. Infection of sugar cane by the nitrogen-fixing bacterium
Acetobacter diazotrophicus. Journal of Experimental Botany 45,
757–766.
RLK in plant–endophytic bacteria association
Kanvinde L, Sastry GRK. 1990. Agrobacterium tumefaciens is
a diazotrophic bacterium. Applied Environmental Microbiology
56, 2087–2092.
Kistner C, Parniske M. 2002. Evolution of signal transduction in
intracellular symbiosis. Trends in Plant Science 7, 511–518.
Krusell L, Madsen LH, Sato S, et al. 2002. Shoot control of root
development and nodulation is mediated by a receptor-like kinase.
Nature 420, 422–426.
Kumar S, Tamura K, Jakobsen IB, Nei M. 2001. MEGA2:
molecular evolutionary genetics analysis software. Bioinformatics
17, 1244–1245.
Kyte J, Doolittle RF. 1982. A simple method for displaying the
hydropathic character of a protein. Journal of Molecular Biology
157, 105–132.
Lange J, Xie Z, Broughton WJ, Vögeli-Lange R, Boller T. 1999.
A gene encoding a receptor-like protein kinase in the roots of
common bean is differentially regulated in response to pathogens,
symbionts and nodulation factors. Plant Science 142, 133–145.
Letunic I, Copley RR, Schmidt S, Ciccarelli FD, Doerks T,
Schultz J, Ponting CP, Bork P. 2004. SMART 4.0: towards
genomic data integration. Nucleic Acids Research Database issue
32, D142–D144.
Li J, Lease KA, Tax FE, Walker JC. 2001. BRS1, a serine
carboxypeptidase, regulates BRI1 signalling in Arabidopsis thaliana. Proceedings of the National Academy of Sciences, USA 98,
5916–5921.
Li J, Wen J, Lease KA, Doke JT, Tax FE, Walker JC. 2002.
BAK1, an Arabidopsis LRR receptor-like kinase, interacts with
BRI1 and modulates brassinosteroid signalling. Cell 110, 213–222.
Logeman J, Schell J, Willmitzer L. 1987. Improved method for the
isolation of RNA from tissues. Analytical Biochemistry 163, 16–20.
Matsubayashi Y, Ogawa M, Morita A, Sakagami Y. 2002. An
LRR receptor kinase involved in perception of a peptide plant
hormone, phytosulfokine. Science 296, 1470–1472.
Matsubayashi Y. 2003. Ligand-receptor pairs in plant peptide
signalling. Journal of Cell Science 116, 3863–3870.
Montoya T, Nomura T, Farrar K, Kaneta T, Yokota T, Bishop
GJ. 2002. Cloning the tomato curl3 gene highlights the putative
dual role of the leucine-rich repeat receptor kinase tBRI1/SR160 in
plant steroid hormone and peptide hormone signalling. The Plant
Cell 14, 3163–3176.
Nishimura R, Hayashi M, Wu GJ, et al. 2002. HAR1 mediates
systemic regulation of symbiotic organ development. Nature 420,
426–429.
Nogueira EM, Vinagre F, Masuda HP, Vargas C, Pádua VLM,
Silva FR, Santos RV, Baldani JI, Ferreira PCG, Hemerly AS.
2001. Expression of sugarcane genes induced by inoculation with
Gluconacetobacter diazotrophicus and Herbaspirillum rubrisubalbicans. Genetics and Molecular Biology 24, 199–206.
Olivares FL, James EK, Baldani JI, Döbereiner J. 1997. Infection
of mottled stripe disease-susceptible and resistant sugarcane
varieties by the endophytic diazotrophs Herbaspirillum. New
Phytologist 135, 723–727.
Pastuglia M, Swarup R, Rocher A, Saindrenan P, Roby D,
Dumas C, Cock JM. 2002. Comparison of the expression patterns
of two small gene families of S gene family receptor kinase genes
during the defence response in Brassica oleracea and Arabidopsis
thaliana. Gene 282, 215–225.
Purdy LH, Liu LJ, Dean JL. 1983. Sugarcane rust, a newly
important disease. Plant Disease 67, 1292–1296.
Radutoiu S, Madsen LH, Madsen EB, et al. 2003. Plant
recognition of symbiotic bacteria requires two LysM receptorlike kinases. Nature 425, 585–592.
Reinhold-Hurek B, Hurek T. 1998. Life in grasses: diazotrophic
endophytes. Trends in Microbiology 6, 139–144.
569
Reis VM, Olivares FL, Döbereiner J. 1994. Improved methodology for isolation of Acetobacter diazotrophicus and confirmation
of its endophythic habitat. World Journal of Microbiology and
Biotechnology 10, 101–104.
Reis VM, de los Santos PE, Tenorio-Salgado S, et al. 2004.
Burkholderia tropicae sp. Nov., a novel nitrogen-fixing, plantassociated bacterium. International Journal of Systems Evolution
Microbiology 54, 1–8.
Ricaud C, Ryan CC. 1989. Leaf scald. In: Ricaud C, Egan BT,
Gillaspie Jr AG, Hughes CG, eds. Diseases of sugarcane.
Amsterdam: Elsevier Science Publishing, Inc., 39–58.
Searle IR, Men AE, Laniya TS, Buzas DM, Iturbe-Ormaetxe I,
Carroll BJ, Gresshoff PM. 2003. Long-distance signalling in
nodulation directed by a CLAVATA1-like receptor kinase.
Science 299, 109–112.
Sevilla M, Burris RH, Gunapala N, Kennedy C. 2001. Comparison of benefit to sugar cane plant growth of an 15N2 incorporation
following inoculation of sterile plants with Acetobacter
diazotrophicus wild-type and Nif- mutant strains. Molecular and
Plant–Microbe Interactions 14, 358–366.
Shiu SH, Bleecker AB. 2001a. Plant receptor-like kinase gene
family: diversity, function, and signaling. Science’s STKE, re22.
Shiu SH, Bleecker AB. 2001b. Receptor-like kinases from Arabidopsis form a monophyletic gene family related to animal receptor
kinases. Proceedings of the National Academy of Sciences, USA
98, 10763–10768.
Shiu SH, Karlowski WM, Pan R, Tzeng Y, Mayer KFX, Li W.
2004. Comparative analysis of the receptor-like kinase family in
arabidopsis and rice. The Plant Cell 16, 1220–1234.
Sikorski MM, Biesiadka J, Kasperska AE, Kopcinska J, Lotocka
B, Golinowski W, Legocki AB. 1999. Expression of genes encoding PR10 class pathogenesis-related proteins is inhibited in
yellow lupine root nodules. Plant Science 149, 125–137.
Stracke S, Kistner C, Yoshida S, et al. 2002. A plant receptor-like
kinase required for both bacterial and fungal symbiosis. Nature
417, 959–962.
Tatusova TA, Madden TL. 1999. Blast 2 sequences: a new tool for
comparing protein and nucleotide sequences. FEMS Microbiology
Letters 174, 247–250.
Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins
DG. 1997. The ClustalX windows interface: flexible strategies for
multiple sequence alignment aided by quality analysis tools.
Nucleic Acids Research 24, 4876–4882.
Urquiaga S, Cruz HS, Boddey RM. 1992. Contribution of nitrogen
fixation to sugarcane: nitrogen-15 and nitrogen balance estimates.
Soil Science Society of America Journal 56, 105–114.
Vargas C, Pádua VLM, Nogueira EM, Vinagre F, Masuda HP,
Silva FR, Baldani JI, Ferreira PCG, Hemerly AS. 2003.
Signalling pathways mediating the association between sugarcane
and endophytic diazotrophic bacteria: a genomic approach.
Symbiosis 35, 159–180.
Von Heijne G. 1991. The signal peptide. Journal of Membrane
Biology 115, 195–201.
Wang Z-Y, Seto H, Fujioka S, Yoshida S, Chory J. 2001. BRI1 is
a critical component of a plasma-membrane receptor for plant
steroids. Nature 410, 380–383.
Wopereis J, Pajuelo E, Dazzo FB, Jiang Q, Gresshoff PM, De
Bruijn FJ, Stougaard J, Szczyglowski K. 2000. Short root
mutant of Lotus japonicus with a dramatically altered symbiotic
phenotype. The Plant Journal 23, 97–114.
Yamamuro C, Ihara Y, Wu X, Noguchi T, Fugioka S, Taktsuto S,
Ashikari M, Kitano H, Matsuoka M. 2000. Loss of function of
a rice brassinosteroid insensitive 1 homolog prevents internode
elongation and bending of the lamina joint. The Plant Cell 12,
1591–1605.