A novel gene, designated as GhNPR1 (Gossypium hirsutum non-expressor of pathogenesis-related genes 1), was isolated from G. hirsutum (cotton) by RT–PCR (reverse transcription–PCR) and RACE (rapid amplification of cDNA ends). The full-length cDNA was 2108 bp long and had an ORF (open reading frame) that putatively encoded a polypeptide of 592 amino acids, with a predicted molecular mass of 66 kDa. Comparison of this protein sequence with that of Arabidopsis thaliana, Brassica juncea and Nicotiana tabacum showed that the amino-acid homology was 52.98, 52.32 and 54.98% respectively. Analysis of the exon–intron structure of the GhNPR1 gene showed that GhNPR1 consisted of four exons and three introns. Southern-blot analysis revealed that the GhNPR1 was a single-copy gene in cotton. Northern-blot analysis indicated that GhNPR1 was constitutively expressed in all tested tissues, including roots, stems and leaves, with the high expression in stems and leaves. In addition, GhNPR1 was also found to be induced by signalling molecules for plant defence responses, such as methyl jasmonate, salicylic acid and ethylene, as well as attack by pathogens, such as Fusarium oxysporum and Xanthomonas campestris. These results suggest that GhNPR1 may play an important role in the response to pathogen infections in cotton plants.

INTRODUCTION

SAR (systemic acquired resistance) is a general plant defence response that can be triggered by a local hypersensitive response to an avirulent pathogen, which renders uninfected parts of the plant resistant to a wide range of normally virulent pathogens [1,2]. SAR is thought to be the consequence of the concerted activation of many genes that are often referred as PR (pathogenesis-related) genes. Although the function of most PR gene products remains to be well defined, some of these proteins have been shown to confer various degrees of pathogen resistance [3,4].

The signalling molecule SA (salicylic acid) has been reported to be essential for the establishment of SAR. Upon the activation of a SAR response, the endogenous level of SA increases dramatically throughout the plant. Exogenous application of SA or its active analogues, such as INA (2,6-dichloroisonicotinic acid) and BTH [benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester], results in the activation of SAR [5,6]. In contrast, removal of SA results in plants that are unable to establish a SAR response and are hypersusceptible to pathogen infections [7,8].

The efforts to genetically interpret the SAR pathway downstream of the SA signal have always been linked to the discovery of numerous alleles of a single gene designated NPR1 (non-expressor of PR genes, also known as NIM1 and SAI1) [3,911]. NPR1 encodes a protein containing two protein–protein interaction domains; BTB/POZ (broad-complex, tramtrack and bric-a-brac/pox virus and zinc finger) and ankyrin repeat [5,12]. Nuclear localization of the NPR1 protein is essential for its proper biological function [13,14]. The NPR1 protein forms an oligomer and is excluded from the nucleus when there is no stimulation. In contrast, a monomeric form is adapted upon SAR stimulation by redoxation, and the nucleus-accumulated NPR1 is assumed to be responsible for the PR gene activation [15]. NPR1 has been shown to be a key component of the SA-regulated PR gene expression and disease-resistance pathway, because NPR1 mutants fail to express PR1, PR2 and PR5, and display enhanced susceptibility to infection even after treatment with SA or INA [3,911]. Furthermore, transgenic Arabidopsis plants overexpressing NPR1 exhibit a more dramatic induction of PR genes upon pathogen infections and exhibit complete resistance to Peronospora parasitica Noco and Pseudomonas syringae pv. maculicola, whereas plants underexpressing NPR1 are more susceptible to these pathogens [16]. In addition, NPR1 is also involved in JA/ET (jasmonate/ethylene)-dependent induced systemic resistance, which is shared by some Pseudomonas fluorescens strains [17], and has been shown to modulate crosstalk between SA- and JA-dependent defence signalling pathways through a mechanism that remains elusive to date [18,19]. It has been reported that the two NPR1-like genes obtained from Arabidopsis, BOP1 (blade-on-petiole 1) and BOP2, function redundantly to control growth asymmetry, an important aspect of patterning in leaves and flowers [20]. Together, these data suggest that other biological functions may be assumed by NPR1 or its analogues derived from different plants.

To our knowledge, the sequence information for the NPR1 proteins of cotton (Gossypium hirsutum) is largely unknown, and no data are available on the NPR1 gene family, gene structure and expression characterization in cotton. In order to reveal the role of NPR1 of cotton plants during pathogen infections, we have begun a series of studies on the NPR1 genes of cotton. In the present study, we have reported the isolation and characterization of a cotton NPR1 homologue, GhNPR1 (G. hirsutum NPR1).

MATERIALS AND METHODS

Plant materials and treatments

The cotton cultivar (G. hirsutum L. cv Lumian 22) was used in the present study. Seeds were surface-sterilized, planted on to water-saturated vermiculite, and grown in a chamber under a 16 h light/8 h dark cycle at 28°C and 80% relative humidity for 2 weeks. For tissue expression analysis at seedling stage, roots, stems and leaves were harvested from the same plants after 14 days of growth and stored at −80°C until use. For tissue expression analysis at budding stage, roots, stems and leaves were harvested from the same plants grown in pots in the greenhouse under standard conditions and stored at −80°C until use. SA, MeJA (methyl JA) and ET were sprayed on to cotton leaves at concentrations of 5 mM, 2 mM and 2 mM respectively. The treated leaves were harvested and immediately frozen in liquid nitrogen, followed by storage at −80°C until use. The infections of cotton by the bacterial pathogen Xanthomonas campestris pv. malvacearum and the fungal pathogen Fusarium oxysporum f. sp. Vasinfectum were performed as described previously [3,10,19].

RNA preparation, cDNA synthesis and DNA extraction

For RNA isolation, the plant tissue samples were collected separately in liquid nitrogen and stored at −80°C until use. Total RNA was extracted with the RNeasy plant mini kit (Qiagen), according to the manufacturer's instructions. Total RNA was first treated with DNase I (Promega) in order to remove potential genomic DNA contamination. Genomic DNA was isolated from seedling leaves using a modified CTAB (hexadecyltrimethylammonium bromide) method, as described by Saghai-Maroof et al. [21]. The quality and concentration of RNA and DNA samples were revealed by ethidium-bromide-stained agarose gel electrophoresis and spectrophotometer analysis.

RNA was used for RT–PCR (reverse transcription–PCR). First-strand cDNA was then synthesized from 2 μg of total RNA by MMLV (Moloney-murine-leukaemia virus) reverse transcriptase (TaKaRa) with an adaptor primer oligo d(T)18 (TaKaRa) at 42°C for 60 min. DNA was subjected to amplification of the genomic DNA sequence of cotton and Southern-blot analysis.

Amplification of GhNPR1 cDNA fragment

To clone the internal conservative fragment, primers P1 and P2 (Table 1) were designed and synthesized (Sangon) on the basis of the conserved amino-acid and nucleotide sequences between AtNPR1 (Arabidopsis thaliana NPR1; accession no. NM105102), BjNPR1 (Brassica juncea NPR1; AY667498) and NtNPR1 (Nicotiana tabacum NPR1; DQ837218). RT–PCR reaction was carried out as follows: pre-denatured at 94°C for 5 min, followed by 35 cycles of amplification (94°C for 50 s, 58°C for 50 s, 72°C for 1 min), and then followed by extension for 5 min at 72°C. The PCR product was purified using gel extraction kit (TaKaRa), ligated to pMD18-T vector (TaKaRa), transformed into Escherichia coli strain DH5α and then sequenced.

Table 1
Primers used in the cloning of full-length cDNA and genomic sequence of GhNPR1 by RACE
PrimerSequenceDescription
P1 5′-GCNGCNATGMGGAARGAGCC −3′ Degenerate primer, forward 
P2 5′-GAACANCGNGGRAARAANCGYTTNCC −3′ Degenerate primer, reverse 
3P1 5′-GCTCAAGTGGATGGAACATCGGAG-3′ 3′-RACE forward primer, outer 
3P2 5′-GTGGACTTGAATGAGGCACCTTTC-3′ 3′-RACE forward primer, nested 
B26 5′-GACTCTAGACGACATCGA(T)17-3′ Universal adaptor primer, outer 
B25 5′-GACTCTAGACGACATCGA-3′ Universal primer, nested 
5P1 5′-CAACCTGTCCTTCGGCGAAGCCTT-3′ 5′-RACE reverse primer, outer 
5P2 5′-GATAGTGAGATCGGATGGTCGTGC-3′ 5′-RACE reverse primer, nested 
AAP 5′-GGCCACGCGTCGACTAGTAC(G)16-3′ Universal adaptor primer, outer 
AUAP 5′-GGCCACGCGTCGACTAGTAC-3′ Universal primer, nested 
ZP1 5′-GCGCGGTACCATCGTTGCTTTCTTCTTCAGG-3′ Full-length cDNA and DNA primer, forward 
ZP2 5′-GCGCGGATCCTTAGCCACTACTACCATTAGC-3′ Full-length cDNA and DNA primer, reverse 
PrimerSequenceDescription
P1 5′-GCNGCNATGMGGAARGAGCC −3′ Degenerate primer, forward 
P2 5′-GAACANCGNGGRAARAANCGYTTNCC −3′ Degenerate primer, reverse 
3P1 5′-GCTCAAGTGGATGGAACATCGGAG-3′ 3′-RACE forward primer, outer 
3P2 5′-GTGGACTTGAATGAGGCACCTTTC-3′ 3′-RACE forward primer, nested 
B26 5′-GACTCTAGACGACATCGA(T)17-3′ Universal adaptor primer, outer 
B25 5′-GACTCTAGACGACATCGA-3′ Universal primer, nested 
5P1 5′-CAACCTGTCCTTCGGCGAAGCCTT-3′ 5′-RACE reverse primer, outer 
5P2 5′-GATAGTGAGATCGGATGGTCGTGC-3′ 5′-RACE reverse primer, nested 
AAP 5′-GGCCACGCGTCGACTAGTAC(G)16-3′ Universal adaptor primer, outer 
AUAP 5′-GGCCACGCGTCGACTAGTAC-3′ Universal primer, nested 
ZP1 5′-GCGCGGTACCATCGTTGCTTTCTTCTTCAGG-3′ Full-length cDNA and DNA primer, forward 
ZP2 5′-GCGCGGATCCTTAGCCACTACTACCATTAGC-3′ Full-length cDNA and DNA primer, reverse 

3′- and 5′-RACE (rapid amplification of cDNA ends) of GhNPR1

On the basis of the sequence of the cloned internal conservative fragments, specific primer 3P1 and the nested specific primer 3P2 (Table 1) were designed and synthesized for 3′-RACE. The first round of PCR was performed with primer 3P1 and universal primer B26. The PCR product was diluted 10-fold for a second round of amplification with the nested primer 3P2 and nested universal primer B25. Both the primary PCR and nested PCR reactions were performed as follows: the cDNA was pre-denatured at 94°C for 5 min followed by 35 cycles of amplification (94°C for 50 s, 52°C for 50 s, 72°C for 1 min); 72°C for 5 min. The product was purified and cloned into pMD18-T vector, followed by sequencing.

For the 5′-ready cDNA cloning, DNA Clean-up System (Promega) was used to purify the first-strand cDNA, and the 5′-end of the purified cDNA was polyadenylated with dCTP by terminal deoxynucleotidyl transferase (TaKaRa). This was followed by ethanol precipitation and resuspension in distilled de-ionized water. Primers 5P1 and 5P2 (Table 1) were designed based on the sequence of the cloned internal fragments. The first round of PCR was performed with AAP (abridged anchor primer) and 5P1. The PCR product was diluted 100-fold for nested PCR with a second round of amplification with AUAP (abridged universal amplification primer) and 5P2. The two steps were carried out under the following conditions: 94°C for 5 min; 35 cycles of amplification (94°C for 50 s, 56°C for 50 s, 72°C for 1 min); extension at 72°C for 5 min. The amplified fragment was electrophoresed, gel extracted and cloned into the pMD18-T vector and sequenced.

Full-length cDNA and genomic sequence amplification of GhNPR1

By comparing and aligning the above three partial fragments with the DNAMAN software, the full-length cDNA of GhNPR1 was deduced. To verify the integrity of this gene, RT–PCR was carried out to amplify nearly the full-length cDNA using the primers ZP1 and ZP2 (Table 1), and the reactions were performed as follows: 94°C for 8 min; 35 cycles of 94°C for 50 s, 58°C for 50 s and 72°C for 3 min; extension at 72°C for 5 min. The purified PCR product was cloned into pMD18-T vector, introduced into E. coli strain DH5α, and the sequence of the construct was verified by sequencing.

Two gene-specific primers, ZP1 and ZP2, designed on the basis of the cDNA sequence, were used to amplify the genomic sequence of GhNPR1 with genomic DNA as template. The reaction was as follows: 94°C for 6 min; 35 cycles of 94°C for 50 s, 55°C for 50 s, and 72°C for 2.5 min; extension at 72°C for 5 min. The PCR product was purified and ligated into pGEM-T easy vector (Promega, USA), transformed into E. coli strain DH5α and then sequenced.

Southern-blot analysis

Genomic DNA (30 μg/sample) was digested for 24 h at 37°C with SacI, EcoRI, HindIII and XbaI, fractionated by 1.0% agarose gel electrophoresis and then transferred on to a Hybond-N+ Nylon membrane (Amersham Pharmacia) using the method described by Sambrook et al. [22]. The gene-specific probe was synthesized with GhNPR1 cDNA by the Primer-a-Gene® labelling system (Promega) according to the manufacturer's instructions. The filters were prehybridized at 65°C for 24 h in 6×SSC, 5×Denhardt's buffer, 0.5% (w/v) SDS and 0.1 mg/ml single-stranded DNA, then hybridized with 32P-labelled probes for 48 h with the same conditions as prehybridization. These filters were washed twice with 2×SSC and 0.1% (w/v) SDS for 15 min each, and washed twice with 1×SSC and 0.1% (w/v) SDS for 15 min. The bands on the membrane was revealed by exposure to X-ray film (Kodak) for 5 days at −80°C.

Northern-blot analysis

Total RNA was extracted with the RNeasy Plant Kit (Qiagen) according to the manufacturer's instructions, and 30 μg total RNA of each sample was separated on 1% formaldehyde agarose gel and blotted on to a Hybond-N+ Nylon membrane. All filters were hybridized with the radiolabelled GhNPR1 cDNA probe. Prehybridization, hybridization and washing of membranes was performed as described above for Southern blotting. The experiments were repeated three times and representative results are presented.

RESULTS

GhNPR1 cloning and sequence analysis

On the basis of the conserved region of plant NPR1, a fragment of approx. 500 bp was amplified with primers P1 and P2. Specific primers designed according to the obtained internal sequence were subsequently used for the amplification of 3′-end (primer 3P1 and B26; 3P2 and B25) and 5′-end (primer 5P1 and AAP; 5P2 and AUAP) cDNA, resulting in a fragment of approx. 500 bp at 3′-end and a fragment of approx. 1200 bp at 5′-end. Finally, the full-length cDNA sequence of NPR1 was deduced and amplified through RT–PCR using the full-length cDNA primers (ZP1 and ZP2), followed by confirmation through sequencing. As shown in Figure 1, the full-length cDNA of G. hirsutum NPR1 (designated as GhNPR1, GenBank accession no. DQ409173) was 2108 bp in size, with a 1766-bp ORF (open reading frame) from nucleotides 163 to 1938 bp, which encoded a protein of 592 amino acids, with a calculated molecular mass of 66 kDa and an isoelectric point of 6.06.

Alignment of the deduced GhNPR1 protein sequence with related NPR1 proteins

Figure 1
Alignment of the deduced GhNPR1 protein sequence with related NPR1 proteins

Amino-acid sequences were aligned for GhNPR1 (DQ409173), BjNPR1 (AY667498), AtNPR1 (NM105102), and NtNPR1 (DQ837218). Black boxes, identical amino acids; grey boxes, similar amino acid residues. Comparison of GhNPR1 and related NPR1 proteins indicated that there were two functional domains: the BTB domain (I) and the ankyrin repeat sequence (II). Nine conserved cysteine residues are indicated by asterisks; these residues may control the oligomerization state. The two crucial cysteine residues at positions 82 and 216 (in AtNPR1) are indicated (▼). Cys521 and Cys529 (in AtNPR1) are also indicated (↓). Two potential NLSs which are used for the nuclear localization are designated by A and B.

Figure 1
Alignment of the deduced GhNPR1 protein sequence with related NPR1 proteins

Amino-acid sequences were aligned for GhNPR1 (DQ409173), BjNPR1 (AY667498), AtNPR1 (NM105102), and NtNPR1 (DQ837218). Black boxes, identical amino acids; grey boxes, similar amino acid residues. Comparison of GhNPR1 and related NPR1 proteins indicated that there were two functional domains: the BTB domain (I) and the ankyrin repeat sequence (II). Nine conserved cysteine residues are indicated by asterisks; these residues may control the oligomerization state. The two crucial cysteine residues at positions 82 and 216 (in AtNPR1) are indicated (▼). Cys521 and Cys529 (in AtNPR1) are also indicated (↓). Two potential NLSs which are used for the nuclear localization are designated by A and B.

The predicted amino acid sequence exhibited 52.98, 52.32 and 54.98% similarity to NPR1 from A. thaliana, B. juncea and N. tabacum respectively. Protein sequence comparison showed that GhNPR1 contains an ankyrin repeat domain and a BTB/POZ domain, both of which are highly conserved among all NPR1 proteins, and are involved in protein–protein interactions [10,12,23]. In addition, a potential bipartite NLS (nuclear localization sequence) was found in the C-terminal 57 amino acids of GhNPR1 (designated as A and B) which were rich in basic residues. NLSs have been shown to be required for nuclear import of NPR1 or NPR1-like proteins in both plants and animals [13].

It is reasonable to expect that the nine cysteine residues (Figure 1, asterisks) that were completely conserved among all the sequences might be involved in the oligomerization modulating and the nuclear localization of NPR1 [19,20]. Specifically, the two cysteine residues (Cys87 and Cys214 in Figure 1) might be crucial for this form of regulation [15]. The completely conserved residue Cys530 was also a potential candidate residue involved in the oxidation of C-terminal cysteine residues, which were required for NPR1 as a co-activator factor [24].

Molecular evolution analysis

In order to study the evolutionary relationships among different NPR1 and NPR1-like proteins from various plant species, a phylogenetic tree was constructed using the DNAMAN software, and all the amino-acid sequences used were derived from GenBank (Figure 2). The results indicated that NPR1 proteins from different plants were more distantly related to those NPR1-like proteins. Among the 16 members of plant NPR1 and NPR1-like proteins, GhNPR1 had the closest association with the NPR1 group, and was more closely related to CpNPR1 (Carica papaya NPR1) than NPR1 from other plant species. We also found that GhNPR1 was highly homologous to NtNPR1, AtNPR1 and OsNPR1 (Oryza sativa NPR1). Studies have shown that the NPR1 can be induced by SA treatment or pathogen infection [19,25], and the NPR1 gene confers resistance to fungi and bacteria in a dose-dependent fashion in transgenic plants, without obvious detrimental effect on plants development [19]. Therefore we predicted that GhNPR1 has the same effect on biotic stress defence, contributing to the positive regulation of SAR.

Phylogenetic relationships of NPR1 and NPR1-like proteins from different species

Figure 2
Phylogenetic relationships of NPR1 and NPR1-like proteins from different species

The tree was generated using the DNAMAN software. All the amino acid sequences of NPR1 and NPR1-like proteins used for construction of the tree are derived from the GenBank database under the following accession numbers: AtNPR1 (NM105102), AtNPR2 (NM118745), AtNPR3 (NM123879) and AtNPR4 (NM118086) from A. thaliana; BjNPR1 (AY667498) from B. juncea; BnNPR1 (AF52717) from Brassica napus; CaNPR1 (DQ648785) from Capsicum annuum; CpNPR1 (AY550242) from Carica papaya; GhNPR1 (DQ409173) from G. hirsutum; MaNPR1a (DQ925843) from Musa acuminata; NtNPR1 (DQ837218) from N. tabacum; OsNPR1 (DQ450948), OsNPR2 (DQ450949), OsNPR3 (DQ450952), OsNPR4 (DQ450954) and OsNPR5 (DQ450956) from O. sativa; StNPR1 (AY615281) from Solanum tuberosum.

Figure 2
Phylogenetic relationships of NPR1 and NPR1-like proteins from different species

The tree was generated using the DNAMAN software. All the amino acid sequences of NPR1 and NPR1-like proteins used for construction of the tree are derived from the GenBank database under the following accession numbers: AtNPR1 (NM105102), AtNPR2 (NM118745), AtNPR3 (NM123879) and AtNPR4 (NM118086) from A. thaliana; BjNPR1 (AY667498) from B. juncea; BnNPR1 (AF52717) from Brassica napus; CaNPR1 (DQ648785) from Capsicum annuum; CpNPR1 (AY550242) from Carica papaya; GhNPR1 (DQ409173) from G. hirsutum; MaNPR1a (DQ925843) from Musa acuminata; NtNPR1 (DQ837218) from N. tabacum; OsNPR1 (DQ450948), OsNPR2 (DQ450949), OsNPR3 (DQ450952), OsNPR4 (DQ450954) and OsNPR5 (DQ450956) from O. sativa; StNPR1 (AY615281) from Solanum tuberosum.

Genomic sequence analysis of GhNPR1

In order to isolate a genomic GhNPR1 clone, the two primers ZP1 and ZP2 were designed on the basis of the sequence of the cDNA and was used in PCR with cotton genomic DNA as template. This produced a 3208-bp genomic fragment, encompassing 2109-bp coding sequence which was interrupted by 1099-bp introns. Comparison between the GhNPR1 genomic (GenBank accession no. EF988657) and cDNA sequence indicated that three introns (534 bp, 163 bp and 401 bp each) were present in the gene. All introns had typical structural characteristics of plant introns, being rich in A+T content (74% for intron 1, 68% for intron 2 and 72% for intron 3), and being typical with regards to splice sites (consensus 5′-GT and AG-3′).

Southern-blot analysis of GhNPR1 in cotton genome

Southern blot was carried out to investigate the genomic organization of the GhNPR1 gene. To avoid cross-hybridization with other NPR1-like genes, the full-length cDNA of GhNPR1 was used as template for probe synthesis. Since the restriction enzymes SacI, EcoRI, HindIII and XbaI had one recognition site on the GhNPR1 sequences, they were used for complete digestion of genomic DNA of cotton. As shown in Figure 3, there were two bands (approx. 13 and 6.2 kb) for SacI digestion, and two bands (approx. 9.2 and 4.1 kb) for Xbal digestion. Although a few bands were detected, only two strong signals (approx. 5.0 and 4.4 kb) for EcoRI digestion, and two strong signals (approx. 7.5 and 3.9 kb) for HindIII digestion. The result implies that there is a single GhNPR1 gene in the cotton genome.

Southern-blot analysis of GhNPR1 in cotton genome

Figure 3
Southern-blot analysis of GhNPR1 in cotton genome

Each lane contains 30 μg of genomic DNA digested with SacI (lane S), EcoRI (lane E), HindIII (lane H) and Xbal (lane X). Following electrophoresis, DNA was transferred on to a nylon membrane and hybridized with the gene-specific probe synthesized with the α-32P-labelled GhNPR1 cDNA.

Figure 3
Southern-blot analysis of GhNPR1 in cotton genome

Each lane contains 30 μg of genomic DNA digested with SacI (lane S), EcoRI (lane E), HindIII (lane H) and Xbal (lane X). Following electrophoresis, DNA was transferred on to a nylon membrane and hybridized with the gene-specific probe synthesized with the α-32P-labelled GhNPR1 cDNA.

Tissue-specific and developmental expression patterns of GhNPR1

To determine the tissue-specific and developmental expression patterns of the GhNPR1 gene, Northern-blot hybridization was performed using total RNA extracted from different organs, which were derived from seedling stage and budding stage. As shown in Figure 4, the signal intensity of the hybridization band for GhNPR1 depended on the developmental stage of the root, stem or leaf investigated. In the seedling stage, the highest amount of GhNPR1 was found in stems, with relatively lower amounts in roots and leaves. In the budding stage, the highest expression pattern of GhNPR1 was seen in leaves, followed by stems, with the lowest in roots.

Northern-blot analysis of the GhNPR1 gene at different stages in roots (R), stems (S) and leaves (L) of cotton

Figure 4
Northern-blot analysis of the GhNPR1 gene at different stages in roots (R), stems (S) and leaves (L) of cotton

Total RNA was prepared from a seedling stage plant (roots, stems and leaves) and budding stage plant (roots, stems and leaves). α-32P-labelled GhNPR1 cDNA was used as probe. Ethidium-bromide-stained rRNA was included as a loading control.

Figure 4
Northern-blot analysis of the GhNPR1 gene at different stages in roots (R), stems (S) and leaves (L) of cotton

Total RNA was prepared from a seedling stage plant (roots, stems and leaves) and budding stage plant (roots, stems and leaves). α-32P-labelled GhNPR1 cDNA was used as probe. Ethidium-bromide-stained rRNA was included as a loading control.

Effect of defence molecules on the transcription level of GhNPR1

It has been shown that the NPR1 can be activated by exogenous SA or its active analogues, such as INA and BTH, resulting in the activation of SAR [15,25,26]. In the present study, we examined gene expression changes of GhNPR1 in response to SA, MeJA and ET. As shown in Figure 5, we found that GhNPR1 mRNA accumulated rapidly and stably compared with the unstressed control plants, and that GhNPR1 gene expression patterns were not affected by these signalling molecules in the seedling stage. Interestingly, the increase in mRNA levels of the plants treated with MeJA was much slower, starting at 12 h of treatment and reaching a maximum after 48 h, compared with those treated with SA and ET.

Northern-blot analysis of the induction of the GhNPR1 gene by 2 mM MeJA (A), 5 mM SA (B) and 2 mM ET (C)

Figure 5
Northern-blot analysis of the induction of the GhNPR1 gene by 2 mM MeJA (A), 5 mM SA (B) and 2 mM ET (C)

Total RNA was isolated at the indicated times after the treatments and was subjected to Northern-blot analysis with α-32P-labelled GhNPR1 cDNA as a probe. Ethidium-bromide-stained rRNA was included as a loading control.

Figure 5
Northern-blot analysis of the induction of the GhNPR1 gene by 2 mM MeJA (A), 5 mM SA (B) and 2 mM ET (C)

Total RNA was isolated at the indicated times after the treatments and was subjected to Northern-blot analysis with α-32P-labelled GhNPR1 cDNA as a probe. Ethidium-bromide-stained rRNA was included as a loading control.

GhNPR1 mRNA accumulation under biotic stress

It has been shown that NPR1 is involved in the resistance response mediated by some R genes [10,11,19]. To gain more direct evidence on whether the GhNPR1 is involved in pathogen attack responses, we inoculated the cotton plants with the fungal pathogen F. oxysporum f. sp. Vasinfectum and the bacterial pathogen X. campestris pv. malvacearum. Northern-blot analysis indicated that the GhNPR1 expression levels were up-regulated in both pathosystems (Figure 6). These results suggest an essential role for GhNPR1 in cotton disease resistance.

Northern-blot analysis of the induction of the GhNPR1 gene by pathogen infections

Figure 6
Northern-blot analysis of the induction of the GhNPR1 gene by pathogen infections

Cotton seedlings (2-week old) were inoculated with F. oxysporum f. sp. Vasinfectum (A) and X. campestris pv. malvacearum (B). The time after inoculation is shown above the blots. α-32P-labelled GhNPR1 cDNA was used as probe. Ethidium-bromide-stained rRNA was included as a loading control.

Figure 6
Northern-blot analysis of the induction of the GhNPR1 gene by pathogen infections

Cotton seedlings (2-week old) were inoculated with F. oxysporum f. sp. Vasinfectum (A) and X. campestris pv. malvacearum (B). The time after inoculation is shown above the blots. α-32P-labelled GhNPR1 cDNA was used as probe. Ethidium-bromide-stained rRNA was included as a loading control.

DISCUSSION

Although the molecular and biochemical characterization of NPR1 or analogous genes in plants has been extensively studied, most are mostly carried out in model plant species, such as Arabidopsis, tobacco and rice. However, NPR1 or its analogues in cotton (Gossypium hirsutum), an economically important plant, have not been reported before. This is the first study to invesigate the existence of NPR1 in cotton and its possible involvement in disease responses. The cloning, characterization and expression of a NPR1 gene from cotton prompt the possibility of a NPR1-based resistance against fungal and bacterial diseases in cotton, which is now under intensive investigation in our laboratory.

Previous studies have shown that NPR1 belongs to a multi-gene family in the genome of many plant species; for example, there were at least six NPR1-like genes in Arabidopsis genome and five genes in rice genome [19,20,27]. In the present study, we have shown that there was a single GhNPR1 gene in the cotton genome (Figure 3). The few weaker bands detected by Southern-blot analysis may represent other NPR1-like genes in cotton genome. However, the number of NPR1-like genes in cotton and the analysis of their functions remain to be investigated in the future.

It has been shown that two conserved cysteine residues (Cys82 and Cys216) are essential for the formation of the AtNPR1 oligomer through disulfide bonds, and that mutations in these residues cause constitutive monomerization and localization of AtNPR1 in the nucleus [15]. In rice, two conserved cysteine residues (Cys76 and Cys216) play an essential role in OsNPR1 oligomer formation [19]. Our experiment indicated that the GhNPR1 protein also contains two conserved cysteine residues at positions 87 and 214 (Figure 1), and the mutation analysis of these residues is currently under investigation to verify their function in the constitutive monomerization and localization of GhNPR1 in the nucleus.

GhNPR1 mRNA was expressed at different levels during different development stages in roots, stems and leaves, indicating that GhNPR1 is regulated over time. During the development of stems and roots, the expression level of GhNPR1 was reduced, whereas GhNPR1 expression increased along with leaf development (Figure 4). This shows that the regulation mechanism of GhNPR1 expression is complex and should be determined further.

NPR1 is expressed at low levels in healthy uninfected plants [25]. Upon pathogen infection or treatment with SA or its functional analogues, the expression of NPR1 is induced by 2–3-fold [5,19,25]. In the present study, the expression pattern of the GhNPR1 was analysed by Northern blotting. The results showed that GhNPR1 could be markedly induced by SA, MeJA and ET (Figure 5), which play important roles in signalling defence responses. Thus it is tempting to speculate that the GhNPR1 is also involved in responses to pathogen infections. To gain more direct evidence, we examined its expression profiles following the inoculation of F. oxysporum f. sp. Vasinfectum and X. campestris pv. malvacearum. The mRNA of GhNPR1 was found to be induced within hours after both pathogen infections (Figure 6). These results suggest that the increased expression of GhNPR1 induced by pathogens and defence signalling molecules may be critical for the activation of the plant defence responses.

Abbreviations

     
  • AAP

    abridged anchor primer

  •  
  • AUAP

    abridged universal amplification primer

  •  
  • At

    Arabidopsis thaliana

  •  
  • Bj

    Brassica juncea

  •  
  • BTB/POZbroad-complex

    tramtrack and bric-a-brac/pox virus and zinc finger

  •  
  • BTH

    benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester

  •  
  • ET

    ethylene

  •  
  • Gh

    Gossypium hirsutum

  •  
  • INA

    2,6-dichloroisonicotinic acid

  •  
  • JA

    jasmonate

  •  
  • MeJA

    methyl JA

  •  
  • NLS

    nuclear localization sequence

  •  
  • NPR

    non-expressor of PR genes

  •  
  • Nt

    Nicotiana tabacum

  •  
  • Os

    Oryza sativa

  •  
  • PR gene

    pathogenesis-related gene

  •  
  • RACE

    rapid amplification of cDNA ends

  •  
  • RT–PCR

    reverse transcription–PCR

  •  
  • SA

    salicylic acid

  •  
  • SAR

    systemic acquired resistance

We thank Dr Xueshui Guo (Department of Biology and Microbiology, South Dakota State University, Brookings, SD, U.S.A.) for critical reading of the manuscript. This work was financially supported in part by the National Natural Science Foundation of China (grant no. 30370928 and 30571215).

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