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. 2011 Dec 29;13(6):579–592. doi: 10.1111/j.1364-3703.2011.00773.x

The arbuscular mycorrhizal symbiosis promotes the systemic induction of regulatory defence‐related genes in rice leaves and confers resistance to pathogen infection

LIDIA CAMPOS‐SORIANO 1, JOSÉ GARCÍA‐MARTÍNEZ 2, BLANCA SAN SEGUNDO 1,✉
PMCID: PMC6638712  PMID: 22212404

SUMMARY

Arbuscular mycorrhizal (AM) symbioses are mutualistic associations between soil fungi and most vascular plants. Their association benefits the host plant by improving nutrition, mainly phosphorus nutrition, and by providing increased capability to cope with adverse conditions. In this study, we investigated the transcriptional changes triggered in rice leaves as a result of AM symbiosis, focusing on the relevance of the plant defence response. We showed that root colonization by the AM fungus Glomus intraradices is accompanied by the systemic induction of genes that play a regulatory role in the host defence response, such as OsNPR1, OsAP2, OsEREBP and OsJAmyb. Genes involved in signal transduction processes (OsDUF26 and OsMPK6) and genes that function in calcium‐mediated signalling processes (OsCBP, OsCaM and OsCML4) are also up‐regulated in leaves of mycorrhizal rice plants in the absence of pathogen infection. In addition, the mycorrhizal rice plants exhibit a stronger induction of defence marker genes [i.e. pathogenesis‐related (PR) genes] in their leaves in response to infection by the blast fungus Magnaporthe oryzae. Evidence indicates that mycorrhizal rice plants show enhanced resistance to the rice blast fungus. Overall, these results suggest that the protective effect of the AM symbiosis in rice plants relies on both the systemic activation of defence regulatory genes in the absence of pathogen challenge and the priming for stronger expression of defence effector genes during pathogen infection. The possible mechanisms involved in the mycorrhiza‐induced resistance to M. oryzae infection are discussed.

INTRODUCTION

Most agricultural crop species all over the world suffer from a vast array of fungal diseases that cause severe yield losses. Rice blast, caused by the fungus Magnaporthe oryzae, is one of the most devastating diseases of cultivated rice (Oryza sativa L.), because of its widespread distribution and destructiveness (Talbot, 2003). The conventional control of rice blast depends on the use of chemically synthesized fungicides. However, the use of such chemicals has several drawbacks, such as their side effects on nontarget organisms, the incidence of development of resistance on prolonged application and the adverse impact on human health and the environment (Yamaguchi and Fujimura, 2005). Breeding for durable resistance to this fungus is a difficult problem, as most of the resistance genes break down in a few years because of their race specificity and the rapid change in pathogenicity of the blast fungus.

Plants have the ability to respond to pathogen infection through a variety of defence reactions (Jones and Dangl, 2006). Pathogens activate basal defence responses in the plant cell through the receptor‐mediated recognition of pathogen/microbe‐associated molecular patterns (PAMPs/MAMPs) and downstream signalling to activate the innate immune response. This innate immune response includes the rapid generation of reactive oxygen species (ROS) and cell wall reinforcement, changes in ion fluxes across the plasma membrane and transient increases in Ca2+ levels, and the activation of protein phosphorylation/dephosphorylation cascades. The production of antimicrobial compounds (i.e. phytoalexins) and the accumulation of pathogenesis‐related (PR) proteins represent a ubiquitous response to pathogen infection in plants (van Loon et al., 2006). The phytohormones salicylic acid (SA), jasmonic acid (JA), ethylene (ET) and abscisic acid (ABA) act as defence signalling molecules (Glazebrook, 2005; Pieterse et al., 2009). The hormone‐mediated signalling pathways do not function independently, but rather influence each other through a complex network of regulatory interactions (Glazebrook, 2005; Pieterse et al., 2009). Depending on the type of plant—pathogen interaction, one or more of these hormone‐regulated signalling pathways tends to predominate. Although the primary mode of interaction between the SA and JA signalling pathways appears to be mutual antagonism, there is also evidence for synergistic interactions between the two hormone‐mediated signalling pathways (Glazebrook, 2005; Pieterse et al., 2009).

Plant resistance responses act not only at the site of pathogen infection, but also at locations remote from this site, referred to as systemic acquired resistance (SAR) (Durrant and Dong, 2004). The establishment of SAR is associated with increased levels of SA, and is characterized by the coordinated expression of a set of PR genes (van Loon et al., 2006; Ward et al., 1991). The regulatory protein NPR1 (nonexpresser of PR genes) has emerged as a master regulator of the SAR response and a transcriptional co‐activator of PR gene expression (Cao et al., 1994; Delaney et al., 1995). NPR1 has been proposed to play a role in the fine tuning of the antagonistic regulation of SA and JA/ET signalling to control gene expression (Leon‐Reyes et al., 2009).

Systemic resistance responses can also be activated by the colonization of roots with certain nonpathogenic microorganisms, such as selected strains of plant growth‐promoting rhizobacteria (PGPR) and fungi (PGPF), a phenomenon known as induced systemic resistance (van Loon et al., 1998; Van der Ent et al., 2009). Although the majority of studies on beneficial microbe‐induced resistance point to a role for JA and ET in the induced systemic resistance response, several examples of PGPR and PGPF that trigger SA‐dependent SAR responses have been documented (Van der Ent et al., 2009).

The arbuscular mycorrhizal (AM) symbiosis is the most widespread symbiotic association formed between plants and fungi (Smith and Read, 2008). Root colonization by AM fungi improves the uptake of water and mineral nutrients, mainly phosphorus and nitrogen, in the host plant in exchange for photosynthetically fixed carbon (Bonfante and Genre, 2010; Parniske, 2008). The AM symbiosis improves plant growth and fitness. Yet another benefit conferred by the AM symbiosis is an improved level of resistance to root‐infecting pathogens (Cordier et al., 1998; Pozo and Azcón‐Aguilar, 2007; Whipps, 2004). Systemic protection to pathogen infection at the root system level has been demonstrated in tomato plants (Cordier et al., 1998; Pozo et al., 2002; Zhu and Yao, 2004). Enhanced resistance to infection by root pathogens may be a consequence not only of improved plant nutrition, but also of competition for root colonization sites or photosynthates, changes in the rhizosphere microbial population or the activation of plant defence mechanisms.

The AM symbiosis may also have an impact on plant interactions with above‐ground pathogens, but the effect of root colonization by AM fungi on foliar pathogens varies depending on the AM fungus and the type of pathogen encountered by the plant. Thus, enhanced resistance or susceptibility to foliar pathogens has been reported in mycorrhizal plants (Fritz et al., 2006; Liu et al., 2007; Shaul et al., 1999; Whipps, 2004). It has been proposed that pathogens with a biotrophic lifestyle, such as powdery mildew and rust fungi, perform better in mycorrhizal plants (Pozo and Azcón‐Aguilar, 2007). With regard to hemibiotrophs, the impact of the symbiosis varies from no effect to a reduction in disease (Lee et al., 2005). Protection against the necrotrophic fungus Alternaria solani has been described in tomato plants (Fritz et al., 2006; de la Noval et al., 2007).

With regard to the effects of AM colonization on host defence gene expression, different responses can be observed in the mycorrhizal roots of different plant species, as the induction and suppression of host defence mechanisms have been described (Dumas‐Gaudot et al., 2000; Gianinazzi‐Pearson et al., 1996; Liu et al., 2007; Pozo et al., 2002). As an example, a systemic repression, or a delay in the activation of PR gene expression, has been reported in leaves of mycorrhizal tobacco plants challenged with Botrytis cinerea, with these plants exhibiting an increase in disease symptom severity (Shaul et al., 1999). Temporal variations in the response of mycorrhizal plants to the challenging pathogen may also occur, the plant defence response being induced during the initial stages of AM symbiosis but subsequently down‐regulated (García‐Garrido and Ocampo, 2002). The determination of how the expression of defence genes is modulated during root colonization by AM fungi, and how this modulation affects the plant—pathogen relationship in above‐ground tissues, is a requisite for a better exploitation of AM fungi in crop protection.

Cereal crops, and rice in particular, are hosts for AM fungi (Campos‐Soriano et al., 2010; Sawers et al., 2008). Recently, we have reported AM‐induced alterations in defence gene expression in the root system of mycorrhizal rice plants (Campos‐Soriano et al., 2010). In this work, our research objectives were twofold: (i) to determine whether the AM symbiosis results in systemic alterations in the expression of defence‐related genes, namely in the leaves of mycorrhizal rice plants; and (ii) to investigate whether the mycorrhizal symbiosis has an impact on resistance to infection by the rice blast fungus M. oryzae. To this end, a rice defensome containing M. oryzae‐regulated genes was initially used to identify genes that are differentially expressed in the leaves of mycorrhizal relative to nonmycorrhizal rice plants. The expression of genes for which a role in the plant defence response is well established was also monitored in the leaves of mycorrhizal plants. Evidence is presented that the AM symbiosis triggers systemic alterations in gene expression and is accompanied by enhanced disease resistance to the foliar pathogen M. oryzae in rice.

RESULTS

Systemic alterations of gene expression in leaves of mycorrhizal rice plants

Rice (O. sativa L. cv. Senia) plants were inoculated with Glomus intraradices (Campos‐Soriano et al., 2010). At 42 days post‐inoculation (dpi), all the events related to fungal development, namely intraradical hyphae, arbuscules at different morphological stages of formation and vesicles, were present in G. intraradices‐inoculated roots, thus confirming the establishment of the symbiotic association. Using microscopic observations of trypan blue‐stained roots, the overall colonization of rice roots ranged from 25% to 30% (results not shown; similar results have been reported previously; Campos‐Soriano et al., 2010).

We investigated whether root colonization by the AM fungus G. intraradices has an effect on the expression of defence‐related responses in leaves of mycorrhizal rice plants. Firstly, gene expression studies were carried out using a rice defensome contained in a macroarray (Campo et al., 2008). The macroarray comprised a total of 6144 expressed sequence tags representing genes whose expression is induced in leaves by M. oryzae infection (at 3, 6, 9, 24 and 48 h post‐inoculation). Differential screening of the rice macroarray was carried out with 33P‐labelled cDNAs obtained from either mycorrhizal plants or mock‐inoculated plants. Only genes showing a statistically significant (P < 0.05) fold change ≥ 2.0 (leaves from mycorrhizal plants vs. leaves from nonmycorrhizal plants) were considered.

Genes up‐regulated in leaves of mycorrhizal plants were grouped into different functional categories (Table 1). Among them were genes involved in defence and stress responses, transcriptional control, signal transduction and protein synthesis. The defence category comprised an important number of genes involved in protection against oxidative stress and the detoxification of ROS. Peroxidases (three genes), catalase, ascorbate peroxidase and thioredoxins were all expressed at higher levels in leaves of mycorrhizal relative to nonmycorrhizal plants. The expression of these genes is typically activated in rice leaves during infection with the rice blast fungus M. oryzae (Campo et al., 2008; Kim et al., 2001; Ramalingam et al., 2003). Three distinct PR genes, namely a PR1, a chitinase and a lipid transfer protein gene, were also included in the defence category (Table 1).

Table 1.

Genes identified as up‐regulated in leaves of mycorrhizal relative to nonmycorrhizal plants, sorted according to functional category. Genes are listed in alphabetical order. Only genes showing a fold change ≥ 2.0 are listed.

Description Accession number* Ratio† P value
1. Defence and stress responses
Acidic endochitinase LOC_Os05g15770 3.49 7.0E‐03
Ascorbate peroxidase (APX8), thylakoid‐bound LOC_Os02g34810 2.51 9.4E‐05
Carbonic anhydrase LOC_Os01g45274 4.51 2.0E‐04
Catalase LOC_Os02g02400 2.24 5.8E‐04
Cathepsin B‐like cysteine proteinase LOC_Os05g24550 2.28 6.8E‐03
Chitinase III LOC_Os01g47070 2.41 1.1E‐04
Glutathione synthetase LOC_Os12g34380 2.01 3.0E‐03
Glycine‐rich protein (GRP0.9) LOC_Os10g31330 3.37 1.1E‐04
Glycine‐rich RNA‐binding protein (GRP1A) LOC_Os03g46770 2.00 3.4E‐03
Glycolate oxidase (GOX) LOC_Os03g57220 3.52 2.7E‐07
Glyoxalase I LOC_Os08g09250 2.56 1.9E‐03
Heat shock 70‐kDa protein 1 (HSC70‐1) LOC_Os11g47760 2.85 3.3E‐05
Lipid transfer protein (LTP11) LOC_Os12g02310 10.24 1.4E‐04
Lipid transfer protein (LTP14) LOC_Os12g02340 4.71 3.1E‐06
Oryzacystatin (cysteine protease inhibitor) LOC_Os01g58890 2.14 4.2E‐03
Oryzain α chain precursor LOC_Os04g55650 9.22 3.0E‐04
Oryzain γ chain precursor LOC_Os09g27030 3.73 2.4E‐04
Pathogenesis‐related protein 1 (PR1) LOC_Os07g03730 2.05 1.9E‐03
Peroxidase (POX8_1) LOC_Os07g48010 2.10 5.0E‐04
Peroxidase 12 precursor LOC_Os01g73200 3.29 3.1E‐04
Peroxidase 71 precursor LOC_Os01g22249 2.37 6.6E‐05
Protein disulphide isomerase (PDI) LOC_Os01g23740 2.22 1.1E‐02
Thioredoxin LOC_Os07g29410 2.33 6.5E‐05
Thioredoxin F2 protein (TRX‐F2) LOC_Os03g07300 5.40 1.7E‐05
Water stress‐induced protein (WSI724) LOC_Os03g45280 2.07 7.9E‐04
2. Transcription
APETALA2 (AP2) domain‐containing transcription factor protein LOC_Os01g04020 2.74 2.4E‐06
Basic helix—loop—helix (bHLH) family protein LOC_Os01g72370 3.98 2.9E‐07
Myb‐related transcriptional activator protein (D13F‐MYBST1) LOC_Os08g04840 3.45 3.2E‐03
Ethylene‐responsive element‐binding protein (EREBP)‐type transcription factor LOC_Os09g26420 4.70 1.7E‐10
Helix—loop—helix DNA‐binding domain containing protein LOC_Os10g40740 5.21 1.3E‐07
DNA‐directed RNA polymerase II subunit LOC_Os07g27930 2.00 2.0E‐03
RNA‐binding protein LOC_Os12g23180 3.52 6.5E‐08
RNA helicase LOC_Os01g43120 2.04 2.3E‐03
RNA helicase LOC_Os09g34910 5.80 1.4E‐02
U1 small nuclear ribonucleoprotein 70 kDa LOC_Os10g02630 9.37 6.7E‐03
Zinc finger transcription factor LOC_Os01g09620 2.00 3.2E‐03
3. Signal transduction
Abscisic stress‐ripening LOC_Os01g73250 4.46 8.9E‐12
Auxin‐repressed protein/auxin‐associated protein LOC_Os03g22270 3.36 2.6E‐07
Auxin‐repressed 12–15‐kDa protein LOC_Os11g44810 3.26 3.4E‐07
Auxin‐responsive protein IAA16 (indoleacetic acid‐induced protein 16) LOC_Os03g53150 3.28 6.6E‐03
BRI1‐KD interacting protein 103 (proton pump interacting protein) LOC_Os09g17730 2.00 4.1E‐03
Brassinolide (BL)‐enhanced gene (OsBLE1) LOC_Os09g38030 2.75 2.8E‐06
Calcium‐binding protein (OsCBP) LOC_Os12g04240 3.40 1.4E‐08
Cation transport protein (chaC) LOC_Os02g26700 2.74 9.4E‐04
GTPase activating‐like protein LOC_Os10g37410 2.00 3.0E‐03
Mitogen‐activated protein kinase (MPK6) LOC_Os10g38950 2.29 2.4E‐04
Phosphatase‐associated family protein SIT4 LOC_Os01g40340 2.76 1.4E‐05
Phosphate transporter LOC_Os12g40340 2.15 3.2E‐04
Ran (Ras‐related GTP‐binding nuclear protein) LOC_Os01g42530 2.00 2.0E‐03
Receptor‐like protein kinase [domain unknown function 26 (DUF26)‐like] LOC_Os03g16950 2.59 6.1E‐05
Remorin 1 LOC_Os04g45070 3.52 3.9E‐08
Serine/threonine protein kinase LOC_Os12g01140 2.09 1.6E‐03
Two‐component response regulator ARR9 (Response reactor 4) LOC_Os01g72330 6.79 9.6E‐14
Vesicle transport v‐SNARE LOC_Os01g51120 2.73 3.1E‐06
4. Protein synthesis, folding and stabilization
30S ribosomal protein 1, chloroplast precursor LOC_Os03g63950 2.16 1.4E‐03
40S ribosomal protein S21 LOC_Os03g22460 7.25 9.8E‐13
40S ribosomal protein S28 LOC_Os10g27174 2.24 1.4E‐03
50S ribosomal protein L13, chloroplast precursor (CL13) LOC_Os01g54540 3.27 1.8E‐03
50S ribosomal protein L15, chloroplast precursor LOC_Os03g12020 5.60 4.5E‐05
Calnexin precursor LOC_Os04g32950 2.80 9.8E‐06
Proteasome subunit β type 5 precursor LOC_Os06g06030 2.95 2.4E‐06
Ribonuclease 3 precursor LOC_Os09g36680 6.28 3.5E‐10
5. Growth and division
Dormancy‐associated protein LOC_Os11g44810 2.47 3.4E‐07
Light‐induced mRNA LOC_Os01g01340 2.49 6.3E‐05
Light‐regulated protein precursor LOC_Os02g16820 2.70 7.2E‐06
6. Energy and metabolism
2‐Oxoisovalerate dehydrogenase α subunit LOC_Os12g08260 2.42 5.1E‐03
Adenyl‐succinate synthetase LOC_Os03g49220 2.07 1.7E‐03
ADP/ATP translocase LOC_Os02g48720 3.00 1.5E‐04
ATP‐dependent Clp protease ATP‐binding subunit clpA homologue LOC_Os12g12850 7.27 4.4E‐16
b‐Keto acyl reductase LOC_Os04g02620 8.45 8.5E‐07
Chlorophyll a/b‐binding apoprotein CP24 precursor LOC_Os04g38410 4.11 2.3E‐09
Chlorophyll a‐b‐binding protein CP29‐1(LHCII protein 4‐1) LOC_Os07g37240 4.22 9.2E‐10
Chlorophyll a/b‐binding protein I precursor LOC_Os09g17740 2.18 9.6E‐03
Chlorophyll a‐b‐binding protein 7 (LHCI type II CAB‐7) LOC_Os07g38960 2.32 6.6E‐05
Chlorophyll a‐b‐binding protein of LHCII type III (CAB) LOC_Os07g37550 2.00 8.0E‐03
Cytochrome P450 LOC_Os10g38110 2.00 3.7E‐03
Cytokinin dehydrogenase precursor LOC_Os01g10110 10.95 1.3E‐02
Dehydroascorbate reductase LOC_Os05g02530 2.11 1.6E‐03
Ferredoxin‐dependent glutamate synthase (Fd‐GOGAT) LOC_Os07g46460 2.22 1.9E‐04
Ferredoxin‐NADP reductase LOC_Os02g01340 2.22 3.1E‐04
Ferredoxin‐NADP reductase LOC_Os06g01850 2.04 1.6E‐03
Fructose‐bisphosphate aldolase LOC_Os11g07020 2.00 4.0E‐03
Glutamine synthetase, catalytic domain LOC_Os04g56400 10.14 1.0E‐02
Glyceraldehyde‐3‐phosphate dehydrogenase LOC_Os08g03290 2.96 4.8E‐04
Glycine dehydrogenase LOC_Os01g51410 2.62 3.8E‐05
Hydrolase, α/β fold family protein LOC_Os03g10620 4.68 2.2E‐03
Hydroxyanthranilate hydroxycinnamoyltransferase LOC_Os04g42250 2.57 1.9E‐05
Magnesium‐protoporphyrin IX monomethyl ester cyclase LOC_Os01g17170 5.33 1.2E‐11
Malate dehydrogenase, glyoxysomal precursor LOC_Os03g56280 2.27 8.1E‐05
NADP‐dependent malic enzyme LOC_Os01g52500 2.00 3.7E‐03
Methyltransferase (NOL1/NOP2/sun family protein) LOC_Os09g29630 3.63 2.2E‐07
Oxygen‐evolving enhancer protein 1 (OEE1) LOC_Os07g37240 2.41 9.2E‐10
Oxygen‐evolving enhancer protein 1 LOC_Os01g31690 2.57 3.1E‐05
Oxygen‐evolving enhancer protein 2 LOC_Os07g04840 13.82 1.9E‐05
Oxygen‐evolving enhancer protein 3‐1 LOC_Os07g36080 3.46 2.7E‐04
Peptide transporter PTR2/nitrate transporter LOC_Os08g05910 2.22 1.2E‐02
Phosphoglycerate kinase LOC_Os05g41640 2.00 4.8E‐03
Phosphoribulokinase precursor LOC_Os02g47020 2.73 2.2E‐05
Photosystem I antenna protein LOC_Os09g01000 4.11 4.7E‐11
Photosystem I antenna protein LOC_Os07g38960 3.62 6.6E‐05
Photosystem I reaction centre subunit III LOC_Os03g56670 5.42 5.8E‐06
Photosystem I reaction centre subunit IV A LOC_Os07g25430 2.56 6.5E‐03
Photosystem I reaction centre subunit V LOC_Os09g30350 3.12 4.9E‐07
Photosystem I reaction centre subunit N LOC_Os12g08770 4.36 1.6E‐04
Photosystem II 10‐kDa polypeptide LOC_Os08g10020 3.15 1.4E‐06
Photosystem II oxygen‐evolving complex protein 2 precursor (PsbP) LOC_Os07g04840 3.82 1.9E‐05
Potassium transporter 5 (AtPOT5) (AtHAK1) (AtHAK5) LOC_Os04g53620 3.59 6.0E‐04
Δ‐12 oleate desaturase LOC_Os02g48560 8.33 1.2E‐08
Pyruvate dehydrogenase E1 component β subunit, mitochondrial precursor (PDHE1‐B) LOC_Os09g33500 2.30 3.7E‐04
Ribulose bisphosphate carboxylase small chain C LOC_Os12g19470 3.84 3.5E‐09
Ribulose bisphosphate carboxylase small chain LOC_Os04g07530 5.59 9.1E‐12
Ribulose bisphosphate carboxylase small chain LOC_Os12g19381 3.69 5.2E‐09
Ribulose bisphosphate carboxylase/oxygenase activase LOC_Os11g47970 3.76 2.5E‐03
Ribulose‐phosphate 3‐epimerase LOC_Os03g07300 2.42 1.7E‐05
S‐Adenosylmethionine decarboxylase 2 LOC_Os02g39795 3.96 8.5E‐10
Serine‐glyoxylate aminotransferase LOC_Os08g39300 2.22 1.7E‐02
Squalene monooxygenase LOC_Os03g12910 2.35 1.7E‐04
Transketolase LOC_Os06g04270 2.00 6.0E‐03
Triosephosphate isomerase LOC_Os09g36450 2.04 1.7E‐03
Type I light‐harvesting chlorophyll a/b‐binding protein of photosystem II (LHCPII) LOC_Os12g17600 2.49 8.7E‐05
Ubiquinol‐cytochrome c reductase complex 8.0‐kDa protein LOC_Os05g33210 5.20 3.3E‐12
7. Unknown function
Ankyrin repeat domain‐containing protein LOC_Os09g33810 5.18 2.0E‐11
CP12 protein precursor LOC_Os01g19740 2.07 1.7E‐03
Expressed protein LOC_Os10g39900 2.00 1.7E‐07
Expressed protein LOC_Os08g14620 5.31 1.0E‐03
Expressed protein LOC_Os03g52680 5.14 5.5E‐07
Expressed protein LOC_Os05g40300 4.06 5.7E‐09
Expressed protein LOC_Os04g48860 3.39 2.9E‐07
Expressed protein LOC_Os02g51020 2.45 6.5E‐03
Expressed protein LOC_Os01g45914 2.28 1.1E‐04
Expressed protein LOC_Os02g45930 2.15 6.1E‐04
Expressed protein LOC_Os02g12480 2.12 2.9E‐03
Expressed protein LOC_Os06g49640 2.78 4.6E‐04
Expressed protein LOC_Os01g48790 4.85 1.3E‐13
Hypothetical protein LOC_Os04g30240 2.50 3.4E‐03
Hypothetical protein LOC_Os01g37790 4.73 5.0E‐03
Hypothetical protein LOC_Os02g42960 3.90 1.8E‐10
Hypothetical protein LOC_Os10g39900 3.05 1.7E‐07
Hypothetical protein, expressed under carbonate stress‐PLN LOC_Os06g19095 3.61 3.9E‐03
Leucine‐rich repeat protein, putative LOC_Os03g32580 2.59 5.6E‐04
Unknown protein LOC_Os10g39900 5.22 1.7E‐07
Unknown protein LOC_Os03g41080 3.04 9.9E‐07
Unknown protein LOC_Os08g08650 2.29 3.8E‐03
Unknown protein LOC_Os12g42250 2.18 3.9E‐04
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Genes validated by real‐time quantitative polymerase chain reaction are indicated in bold type.

*

Locus ID: locus name from the Rice Genome Annotation Project (http://rice.plantbiology.msu.edu).

†

Fold change: signal intensity ratio of leaves of mycorrhizal plants relative to leaves of nonmycorrhizal plants.

Of interest, the macroarray data revealed the up‐regulation of genes encoding distinct transcription factors, such as members of the AP2/EREBP (APETALA2/ethylene‐responsive element‐binding protein) family of transcription factors (OsAP2, Os01g04020 and OsEREBP, Os09g26420) and a basic helix—loop—helix (bHLH)‐type transcription factor (OsbHLH, Os01g72370) (Table 1). The functional category of signal transduction comprised genes encoding the receptor‐like protein kinase DUF26 (domain unknown function 26) (OsDUF26, Os03g16950), a remorin1 (Os04g45070), a calcium‐binding protein (OsCBP, Os12g04240) and several protein kinases. The MPK6 (mitogen‐activated protein kinase 6, OsMPK6, Os10g38950) gene was also found to be up‐regulated in the leaves of mycorrhizal plants.

Several genes identified by macroarray analysis were randomly selected, and their expression was analysed further by quantitative reverse transcriptase‐polymerase chain reaction (RT‐qPCR). This analysis confirmed the up‐regulation of transcription factor genes, namely OsAP2, OsEREBP and OsbHLH (Fig. 1A), as well as genes involved in signal transduction processes, namely OsDUF26 and OsMPK6 (Fig. 1B) (*P ≤ 0.05 and **P ≤ 0.001). The induction of various OsDUF26 genes in the rice—M. oryzae interaction has been described previously (Campo et al., 2008; Han et al., 2004; 2004, 2009). A role for OsMPK6 in the rice defence response to pathogen infection has been reported (Shen et al., 2010).

Figure 1.

Figure 1

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Changes in transcript abundance of transcription factor genes and genes involved in signal transduction processes in leaves of mycorrhizal rice plants. The expression of genes selected by macroarray analysis was determined by quantitative reverse transcriptase‐polymerase chain reaction (RT‐qPCR). Leaves were collected from mycorrhizal plants at 42 days post‐inoculation (dpi) with the arbuscular mycorrhizal (AM) fungus Glomus intraradices (+Gi) or from mock‐inoculated plants (−Gi). To normalize the RT‐qPCR data, each gene was compared with the ubiquitin 1 (OsUbi1, Os06g46770) transcript. Three independent biological samples were analysed with similar results. Error bars indicate the standard error (SE) from three technical replicates. Asterisks indicate a significant difference between conditions (*P ≤ 0.05 and **P ≤ 0.001). (A) Expression of the transcription factor genes OsAP2 (Os01g04020), OsEREBP (Os09g26420) and OsbHLH (Os01g72370). (B) Expression of the receptor like‐protein kinase OsDUF26 (Os03g16950) and MPK6 (Os10g38950) genes. AP2, APETALA2; bHLH, basic helix—loop—helix; DUF26, domain unknown function 26; EREBP, ethylene‐responsive element‐binding protein; MPK6, mitogen‐activated protein kinase 6.

Overall, gene expression studies revealed that the AM symbiosis is accompanied by systemic alterations in gene expression in rice leaves. Among the mycorrhiza‐induced genes were genes functioning in protection against oxidative stress, transcriptional control of gene expression and signal transduction processes.

Systemic activation of regulatory genes of the host defence response in mycorrhizal rice

The expression of genes playing a regulatory role in the plant defence response, whose expression is also known to be activated by M. oryzae infection in rice leaves, was monitored in leaves of mycorrhizal and nonmycorrhizal plants. These included: OsNPR1 (Os01g09800), OsJAmyb (JA‐regulated myb transcription factor, Os11g45740) and OsMPK7 (mitogen‐activated protein kinase 7, Os05g49140) (Gómez‐Ariza et al., 2007; Lee et al., 2001; Reyna and Yang, 2006). Whereas OsNPR1 functions in the SA‐mediated signalling pathway for the activation of defence responses, OsJAmyb is involved in the JA‐mediated signalling pathway (Lee et al., 2001). OsMPK7 was also found to be highly induced by JA treatment in rice leaves (Reyna and Yang, 2006). RT‐qPCR analysis demonstrated that all three genes, OsNPR1, OsJAmyb and OsMPK7, are expressed at significantly higher levels in leaves of mycorrhizal relative to nonmycorrhizal plants (Fig. 2A; *P ≤ 0.05 and **P ≤ 0.001). Equally, RT‐qPCR revealed that the allene oxide cyclase (OsAOC, Os03g32314) gene is highly expressed in leaves of mycorrhizal relative to nonmycorrhizal plants (Fig. 2B; P ≤ 0.001). The AOC enzyme is involved in the synthesis of 12‐oxo‐phytodienoic acid (OPDA), the ultimate precursor of JA (Schaller, 2001).

Figure 2.

Figure 2

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Expression of regulatory defence‐related genes and genes acting in Ca2+‐mediated signalling processes in mycorrhizal rice plants. Gene expression was determined by quantitative reverse transcriptase‐polymerase chain reaction (RT‐qPCR) in leaves from mycorrhizal (+Gi) and nonmycorrhizal (−Gi) plants. Expression levels were normalized using the rice cyclophilin gene (OsCyc, Os02g02890). Similar results were obtained using the ubiquitin 1 (OsUbi1) gene to normalize the expression data. Three independent biological samples were analysed with similar results. Error bars indicate the standard error from three technical replicates. Asterisks indicate a significant difference between conditions (*P ≤ 0.05 and **P ≤ 0.001). (A) Analysis of OsNPR1 (Os01g09800), OsJAmyb (Os11g45740) and OsMPK7 (Os05g49140) gene expression. (B) Expression of the OsAOC gene (Os03g32314) involved in jasmonic acid (JA) biosynthesis. (C) Expression of rice genes acting in Ca2+‐mediated signalling processes: OsCBP (Os12g04240), OsCaM (Os04g36660) and OsCML4 (Os03g53200). Amyb, JA‐regulated myb transcription factor; AOC, allene oxide cyclase; CaM, calmodulin; CBP, calcium‐binding protein; CML4, calmodulin‐like 4; MPK7, mitogen‐activated protein kinase 7; NPR1, nonexpresser of PR1.

From these results, it appears that genes playing a regulatory role in the rice response to pathogen infection, which function in either an SA‐dependent (OsNPR1) or JA‐dependent (OsJAmyb and OsMPK7) manner, as well as the OsAOC gene involved in JA biosynthesis, are all up‐regulated in the leaves of mycorrhizal rice plants.

Systemic activation of genes involved in Ca2+‐mediated signalling processes in mycorrhizal rice

The crucial role of calcium as a second messenger in plant innate immunity is well established (Lecourieux et al., 2006). Cellular Ca2+ signals are decoded and transmitted by CBPs that relay this information into downstream responses. In line with this, the OsCBP (Os12g04240) gene encoding a CBP from rice was identified among the set of genes overexpressed in the leaves of mycorrhizal plants (Table 1). In other studies, the activation of CBP genes during the infection of rice leaves with M. oryzae has also been reported (Kim et al., 2001; Shim et al., 2004).

In this work, the AM‐induced systemic alterations in the expression of genes acting in Ca2+‐mediated signalling processes were further explored. Initially, the up‐regulation of the OsCBP gene identified by macroarray analysis was confirmed by RT‐qPCR analysis (Fig. 2C; P ≤ 0.05). Next, we investigated the expression of the calmodulin (OsCaM, Os04g36660) and calmodulin‐like 4 (OsCML4, Os03g53200) genes in mycorrhizal rice. OsCaM and OsCML4 expression is activated in response to infection by the rice blast fungus, as recorded in gene expression databases [Rice Expression Database (RED) http://cdna02.dna.affrc.go.jp/RED/ and ‘Genevestigator’https://www.genevestigator.com/gv/). As shown in Fig. 2C, a tendency for a higher expression of the two genes, OsCaM and OsCML4, occurs in leaves of mycorrhizal relative to nonmycorrhizal plants.

Although not proven, the observed up‐regulation of genes encoding different types of calcium sensor protein (OsCBP, OsCaM and OsCML4) in mycorrhizal plants points to a possible role of Ca2+‐mediated signalling processes in the AM‐induced systemic resistance to pathogen infection in rice plants.

Effect of the AM symbiosis on resistance to the rice blast fungus M. oryzae

We then addressed the very important question of whether mycorrhizal rice is resistant to infection by a foliar pathogen, namely the rice blast fungus. Blast resistance assays were conducted on young leaves of rice plants at 42 dpi with G. intraradices. As a control, young leaves from mock‐inoculated plants were assayed. Leaves were inoculated with increasing doses of M. oryzae spore suspension, as described previously (Coca et al., 2004). Using a concentration of inoculum of 106 spores/mL, leaves from mock‐inoculated plants developed clear symptoms of infection at 2–3 dpi (Fig. 3A, −Gi). By this time, small necrotic spots were also evident on M. oryzae‐infected leaves of mycorrhizal plants. Lesions developed actively with time on the leaves of nonmycorrhizal plants, these leaves being severely damaged at 7 dpi with M. oryzae spores (Fig. 3A, −Gi). Under the same experimental conditions, however, leaves of mycorrhizal plants showed a clear reduction in disease severity (Fig. 3A, +Gi).

Figure 3.

Figure 3

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Resistance to infection by the rice blast fungus Magnaporthe oryzae in mycorrhizal rice plants. Leaves were collected from mycorrhizal plants at 42 days post‐inoculation (dpi) with the arbuscular mycorrhizal (AM) fungus Glomus intraradices (+Gi) or from mock‐inoculated plants (−Gi). Results shown are from one of three experiments that produced similar results. (A) Leaves were challenged with M. oryzae spores using the detached leaf assay (Coca et al., 2004). Increasing doses of M. oryzae (PR9 strain) spores were used for inoculation (106, 105 and 104 spores/mL). Control leaves were inoculated with sterile water. Disease symptoms at 3, 5 and 7 dpi of leaves are shown. (B) Percentage of leaf area affected by blast lesions in leaves of nonmycorrhizal and mycorrhizal rice plants at 3, 5 and 7 dpi with M. oryzae spores. The percentage of lesion area was determined using Image Analysis Software, Assess 2.0, for plant disease quantification (Lamari, 2008). Bars represent the average of three independent experiments. For each experiment, at least four leaves were inoculated with each inoculum and five inoculations per leaf were made. Means and standard errors are indicated. Bars with different letters are significantly different by P ≤ 0.05.

We further tested the ability of mycorrhizal plants to block the growth of M. oryzae in leaf tissues by determining the percentage of the leaf area affected by blast lesions. A direct correlation between lesion area and inoculum concentration was observed in fungus‐infected leaves of nonmycorrhizal plants, these lesions gradually increasing with time (Fig. 3B). In agreement with visual inspection, the percentage of lesion area was higher in M. oryzae‐infected leaves of nonmycorrhizal plants relative to mycorrhizal plants. Differences were more evident at 7 dpi with the highest inoculum dose. Lesion development was apparently blocked in fungus‐inoculated leaves of mycorrhizal rice plants. Finally, fungal sporulation did not occur in the leaves of mycorrhizal rice, not even at 7 dpi with the highest inoculum dose used in this study (106 spores/mL), as revealed by spore counting (results not shown).

Knowing that mycorrhizal plants exhibit a resistance phenotype to the rice blast fungus, it was of interest to examine PR gene expression in the leaves of these plants. PR genes whose expression has been reported to be activated by M. oryzae infection in rice leaves were assayed (Gómez‐Ariza et al., 2007; Quilis et al., 2008). These included: OsPR1a (Os07g03710; a member of the PR1 family different from that identified by macroarray hybridization, Table 1), OsPR5 (thaumatin‐like protein, Os03g46070), OsPR10 (Os03g18850) and OsPBZ1 (probenazole inducible 1, Os12g36880). The PR1 and PBZ1 genes are typical markers of the activation of the rice defence response to pathogen infection (Agrawal et al., 2001; Midoh and Iwata, 1996). PR gene expression was initially analysed in noninfected leaves from both mycorrhizal and nonmycorrhizal plants. Intriguingly, reduced levels of transcript accumulation were observed for all four genes (OsPR1a, OsPR5, OsPBZ1 and OsPR10) in the leaves of mycorrhizal relative to nonmycorrhizal plants in the absence of pathogen infection (Fig. 4, 0 dpi). In other studies in leaves of Medicago and tomato plants, the down‐regulation of PR gene expression as a result of AM symbiosis has also been described (Fiorilli et al., 2009; Liu et al., 2007).

Figure 4.

Figure 4

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Expression of pathogenesis‐related (PR) genes in leaves of nonmycorrhizal and mycorrhizal rice plants (grey and black bars, respectively) in response to Magnaporthe oryzae infection. For inoculation, a suspension of M. oryzae spores at 1 × 106 spores/mL was used. Total RNA was isolated at the indicated times after inoculation with M. oryzae spores (0, 1 and 3 days). Changes in transcript abundance were determined by quantitative reverse transcriptase‐polymerase chain reaction (RT‐qPCR) analysis. The relative expression of the OsPR1a (Os07g03710), OsPR5 (Os03g46070), OsPBZ1 (Os12g36880) and OsPR10 (Os03g18850) genes is shown. The gene expression level was normalized using the rice cyclophilin gene (OsCyc, Os02g02890) as an internal reference. Similar results were obtained using the ubiquitin 1 (OsUbi1) gene as an internal reference. Three independent biological samples were analysed with similar results. Error bars indicate the standard error from three technical replicates. Asterisks indicate a significant difference between conditions (*P ≤ 0.05 and **P ≤ 0.001). PBZ1, probenazole inducible 1.

PR gene expression was then monitored in M. oryzae‐infected leaves of both mycorrhizal and nonmycorrhizal plants at two different times after inoculation with M. oryzae spores (1 and 3 dpi) (Fig. 4, 1 dpi, 3 dpi). Under pathogen infection conditions, a potentiation of PR gene expression was observed. In the case of the OsPR1a, OsPR5 and OsPBZ1 genes, a significantly higher expression occurred in the leaves of mycorrhizal plants at 3 dpi with M. oryzae spores (Fig. 4, 3 dpi; *P ≤ 0.05 and **P ≤ 0.001). With regard to OsPR10, a stronger activation occurred at 1 dpi with M. oryzae in leaves of mycorrhizal rice (Fig. 4, P ≤ 0.05).

Overall, the results presented here suggest that the AM symbiosis confers systemic resistance to infection by the rice blast fungus M. oryzae. Consistent with the observed resistance phenotype, a stronger induction of PR gene expression occurs in leaves of mycorrhizal plants in response to infection with the rice blast fungus.

DISCUSSION

AM fungi are mainly thought to facilitate nutrient uptake in plants, an aspect that has received great interest in AM research, although AM fungi can perform several other functions that are equally beneficial for the plant. Evidence indicates that root colonization by the AM fungus confers resistance to soil‐borne pathogens (Pozo et al., 2010). Priming of defence reactions has been proposed to mediate the mycorrhiza‐induced resistance at the root system level (Pozo et al., 2009). As an example, Cordier et al. (1998) demonstrated a rapid and strong activation of defence mechanisms in nonmycorrhizal parts of Glomus mossae‐inoculated tomato roots, which was accompanied by resistance to Phytophthora parasitica infection in these roots.

However, the effect of the AM symbiosis during pathogen infection in above‐ground tissues is variable, and mycorrhizal colonization does not always increase disease resistance to foliar pathogens. Although systemic resistance to leaf pathogens induced by root colonization has been reported (Liu et al., 2007), there are also examples in which mycorrhizal colonization results in increased susceptibility to infection by biotrophic pathogens in aerial parts of the plant (Gernns et al., 2001; Pozo and Azcón‐Aguilar, 2007; Whipps, 2004). A differential pathogen resistance is sometimes observed depending on the AM fungus (de la Noval et al., 2007). Whenever AM‐induced bioprotective effects against foliar pathogens are observed, the molecular mechanisms responsible for this effect are still poorly understood.

Studies in Medicago have shown that the AM symbiosis is accompanied by alterations in gene expression in root and shoot tissues, and that these AM‐regulated transcriptional responses differ between the two tissues (Liu et al., 2007). Consistent with the results presented here in the leaves of mycorrhizal rice plants, genes involved in transcriptional regulation (e.g. AP2/EREBP transcription factor) and in Ca2+‐mediated signalling processes are also induced in shoots of mycorrhizal Medicago plants (Liu et al., 2007). Moreover, the down‐regulation of PR gene expression is also observed in leaves of Medicago mycorrhizal plants in the absence of pathogen infection (Liu et al., 2007). Thus, our results on the systemic alterations in gene expression in mycorrhizal rice plants are essentially in agreement with those reported by Liu et al. (2007) in mycorrhizal Medicago plants. Root colonization by G. intraradices results in enhanced disease resistance in both rice and Medicago plants.

An interesting aspect of this study comes from the observation that the AM symbiosis is accompanied by systemic alterations in the expression of genes playing a regulatory role in the host immune system. It should be mentioned here that little attention has been paid to the study of defence regulatory genes during symbiotic interactions, and most studies have focused on the mycorrhiza‐regulated expression of defence effector genes, such as PR genes. Evidence is presented here on the systemic activation of OsNPR1, OsJAmyb and OsMPKs in the leaves of mycorrhizal rice plants. With regard to NPR1 protein functioning, it is well known that pathogen recognition triggers alterations in the cellular redox status which, in turn, results in changes in the NPR1 protein. The inactive cytosolic oligomeric form of NPR1 is reduced to a monomeric state that translocates to the nucleus for the activation of PR gene expression (Kinkema et al., 2000; Mou et al., 2003). Although most studies on NPR1 functioning have been performed in Arabidopsis, the existence of a disease resistance pathway in rice similar to the Arabidopsis NPR1‐mediated signalling pathway has been described (Chern et al., 2001; Quilis et al., 2008). Intriguingly, even though OsNPR1 expression is up‐regulated in leaves of mycorrhizal rice, its transcriptional activation is not accompanied by an enhanced expression of the downstream NPR1‐regulated rice PR genes (i.e. PBZ1, PR5) (Quilis et al., 2008). Instead, PR gene expression appears to be lower in leaves of mycorrhizal relative to nonmycorrhizal plants in the absence of pathogen challenge. Only under pathogen infection conditions are the NPR1‐regulated PR genes expressed at higher levels in the leaves of mycorrhizal rice plants. Assuming that the activation of OsNPR1 expression occurs first, and that this is followed by translation of the mycorrhiza‐induced OsNPR1 transcripts, there is the possibility that the observed systemic transcriptional activation of OsNPR1 leads to the accumulation of inactive NPR1 proteins. This would explain why the activation of PR gene expression does not occur in the leaves of mycorrhizal plants, even though OsNPR1 expression is activated in these leaves. The OsNPR1 protein will only become active under pathogen infection conditions. Together with this, the pool of latent OsNPR1 proteins will respond more rapidly and/or more strongly to pathogen challenge which, in turn, will result in the strong expression of PR genes and enhanced disease resistance. In favour of these hypotheses, several transcription factors or MPK proteins have been shown to accumulate in an inactive state in a subcellular compartment different from that in which these regulatory proteins exert their activity during rhizobacteria‐ or chemically induced systemic resistance (Beckers et al., 2009; Dahan et al., 2010). Regulation by the AM symbiosis of the cellular redox status may contribute to the maintenance of the inactive oligomeric NPR1 protein in the leaves of mycorrhizal rice plants. Clearly, these strategies provide the plant with an enhanced capacity to effectively counteract pathogen infection, and are less costly than the constitutive expression and massive accumulation of a battery of defence effector proteins in systemic tissues. Further studies are, however, needed to determine whether there is a pool of inactive NPR1 proteins in the leaves of mycorrhizal rice in the absence of pathogen infection.

Our current knowledge of the role of the defence‐related hormones, SA, JA and ET, during the development of systemic responses, SAR and induced systemic resistance comes mainly from studies in dicotyledonous species. In certain species, such as Solanum lycopersicum, Lotus japonicus, Glycine max and Cucumis sativus, the defence‐related JA (and its jasmonate derivatives) appears to control defence gene expression during the AM symbiosis (Hause and Schaarschmidt, 2009; López‐Ráez et al., 2010). In other species, however, JA does not appear to play a role during the AM symbiosis (Riedel et al., 2008). Our study showed that the AM symbiosis results in the systemic activation of genes functioning in the SA‐dependent (OsNPR1) and JA‐dependent (OsJAmyb and OsMPK7) signalling pathways. The expression of the OsAOC gene, which is involved in JA synthesis, was also found to be activated in leaves of mycorrhizal rice. These findings raise the possibility of a possible mycorrhiza‐regulated modulation of the level of the defence‐related hormones SA and/or JA in rice plants, an aspect that remains to be investigated.

It is generally assumed that pathogens that require a living host (biotrophs) are more sensitive to SA‐mediated responses, whereas pathogens that kill the host (necrotrophs) are generally affected by JA/ET‐mediated defences (Glazebrook, 2005). The effectiveness of the mycorrhiza‐induced resistance in systemic tissues may well depend, at least in part, on the lifestyle of the pathogen attacking above‐ground tissues and/or the signalling pathway(s) that is activated by each particular pathogen in the host plant. The fungus M. oryzae is a hemibiotrophic pathogen which maintains an initial biotrophic phase with the host, and then switches to a necrotrophic lifestyle (Campos‐Soriano and San Segundo, 2009; Talbot, 2003). Hence, the mycorrhiza‐induced bioprotective effect to infection by this hemibiotrophic pathogen may operate in a different manner, or at a different level, depending on whether the fungus invades the host tissue in a biotrophic or necrotrophic manner. From our results, it appears that, under severe infection conditions (105–106 spores/mL), the fungus manages to develop small lesions in the leaves of mycorrhizal rice plants early during the infection process. However, leaf disease progression appears to be blocked at later stages of the infection process, thus suggesting a bioprotective effect of the AM symbiosis during the necrotrophic period of leaf colonization by M. oryzae. A better knowledge of the roles of the SA and JA defence hormones in the rice—M. oryzae pathosystem is, however, needed to better understand the involvement of these defence signalling pathways in the mycorrhiza‐induced resistance to M. oryzae infection in rice. It would also be of interest to determine whether the mycorrhiza‐induced systemic alterations in host gene expression have an impact on diseases caused by other types of pathogens in rice plants, both necrotrophic and biotrophic pathogens.

Finally, it is well known that Ca2+ signalling mediates the recognition of both pathogenic and beneficial microbes in plants. Thus, rapid and transient elevations in cellular Ca2+ concentration occur during PAMP‐induced defence responses (Blume et al., 2000), as well as during the AM symbiosis in roots (Bonfante and Genre, 2010). Specificity in the Ca2+ signalling system and downstream responses most probably depends on the availability of a specific set of Ca2+ sensors (and their target proteins) in each type of interaction. Our results show distinct calmodulin, calmodulin‐like protein and calmodulin‐binding protein genes whose expression is up‐regulated in the leaves of mycorrhizal rice plants; these genes are also up‐regulated by M. oryzae infection. As calmodulin and calmodulin‐related proteins are known to function as sensors of Ca2+ signals in pathogenic interactions, these particular genes may be involved in the decoding of Ca2+ signals induced by M. oryzae infection in the leaves of mycorrhizal rice.

To summarize, the results presented here suggest that the AM symbiosis confers resistance to infection by the rice blast fungus in rice plants. The protective effect of the AM symbiosis is accompanied by the systemic activation of genes playing a regulatory role in host immunity. Priming for a faster and/or stronger PR gene expression also occurs in leaves of mycorrhizal rice following infection by the rice blast fungus M. oryzae. Overall, this study provides new insights into the molecular mechanisms involved in the mycorrhiza‐induced resistance to M. oryzae infection in rice plants, which might be useful to better exploit the benefits of the AM symbiosis in rice protection under field conditions.

EXPERIMENTAL PROCEDURES

Plant and fungal material

The elite japonica rice cultivar Senia was used in this study. Rice plants were soil grown at 27 ± 2 °C under an 18‐h/6‐h light/dark photoperiod. The AM fungus G. intraradices (DAOM197198) was aseptically grown on Petri dishes containing modified minimal medium agar at 24 °C until the fungus sporulated (approximately 3 months). The monoxenic culture (G. intraradices and carrot roots) was grown as described previously (Campos‐Soriano et al., 2010).

The M. oryzae isolate PR9, which establishes a compatible interaction with the commercial rice cultivar Senia, was used for blast disease resistance assays (Coca et al., 2004). The fungus was grown for 15 days at 28 °C on oat agar (Oatmeal Agar, Difco, France) Petri dishes. Spores were collected from fungal mycelium by adding sterile water to the surface of the mycelium. After filtration and microscopic observation, spores were adjusted to the appropriate concentration with sterile water with a Bürker counting chamber.

Inoculation of rice plants with the AM fungus G. intraradices

Each plant was grown in a separate pot on a sand—vermiculite mixture (50:50, v/v) and inoculated with a piece of monoxenic culture of G. intraradices (Campos‐Soriano et al., 2010). Control plants were mock‐inoculated with uninfected carrot roots. Plants were watered every 2 days with a modified Hoagland nutrient solution containing 25% of the standard phosphorus concentration to induce the establishment of symbiosis. Plants were harvested at 42 dpi; their roots were extensively washed with sterile water and analysed for AM colonization. Microscopic analysis of trypan blue‐stained roots was performed to confirm the establishment of the AM symbiosis, as described previously (Campos‐Soriano et al., 2010). Three separate experiments using at least three plants and conditions (noninoculated and G. intraradices‐inoculated) were carried out.

RNA isolation and analysis of gene expression by RT‐qPCR

Total RNA was isolated from rice leaves using the TRIZOL® Reagent (Invitrogen, Carlsbad, CA, USA). For gene expression studies, young leaves from mycorrhizal rice plants were collected and immediately frozen in liquid nitrogen. Leaf material was also harvested from nonmycorrhizal plants. At least six leaves from three individual plants were analysed. RT‐qPCR analyses were carried out in 96‐well optical plates in a LightCycler® 480 Real‐Time PCR System (Roche, Mannheim, Germany) according to the following programme: 10 min at 95 °C, followed by 45 cycles of 95 °C for 10 s, 60 °C for 30 s, and an additional cycle of dissociation curves to ensure a unique amplification. The reaction mixture contained 10 µL of 2 × SYBR Green Master mix reagent (Roche), 2 µL of cDNA sample and 300 µm of each gene‐specific primer in a final volume of 20 µL. Primers used for RT‐qPCR are shown in Table S1 (see Supporting Information). Three replicate reactions were used for each sample. Relative quantification of specific mRNA levels was performed using the comparative 2−Δ(ΔCt) method (Livak and Schmittgen, 2001). As internal controls, ubiquitin 1 (OsUbi1) and cyclophilin (OsCyc) were routinely used. Three independent biological samples were analysed. One‐way analyses of variance (anovas) were used to evaluate differences in gene expression between mycorrhizal and nonmycorrhizal plants. For anovas in which the F test was significant at 0.05 or a lower probability level, the least‐significant difference test was applied to detect differences.

Macroarray hybridization, scanning and data analysis

A macroarray containing genes that are up‐regulated during M. oryzae infection in rice leaves was used (Campo et al., 2008). For hybridization experiments, cDNAs were obtained by reverse transcription from 40 µg of total RNA and labelled with 33P‐α‐deoxycytidine triphosphate (dCTP) using SuperScript II Reverse Transcriptase (Invitrogen). The labelled cDNA probes were denatured at 100 °C for 5 min, followed by 5 min on ice, and then used for hybridization. Before hybridization, the membranes were washed in 0.5% sodium dodecylsulphate (SDS) for 30 min at 80 °C. The membranes were prehybridized in 5 × standard saline citrate (SSC), 0.5% SDS and 5 × Denhardt's solution at 65 °C for at least 1 h. Hybridization was carried out for 40–64 h at 60 °C. The membranes were washed at 65 °C in 2 × SSC, 0.1% SDS for 20 min, and twice in 0.2 × SSC, 0.1% SDS for 30 min. Hybridization signals were recorded by phosphor imaging (FujiFilm FLA3000 Phosphorimager, Berlin, Germany). The image data obtained were imported into the software program ArrayVision 7.0 (Imaging Research, St. Catharines, ON, Canada) for spot detection and quantification of hybridization signals. Backgrounds were subtracted using ArrayVision 7.0 to obtain raw signal intensities. To control against biological errors, hybridizations were performed with two independently labelled cDNAs from RNA samples obtained from two independently pooled leaf samples. To assess the reproducibility of the macroarray analysis, two independent hybridizations were conducted for each sample and each biological replicate was hybridized in a different membrane. Normalization and statistical validation were carried out using ArrayStat 1.0 software (Imaging Research). In this software, normalization is performed in two steps: first, an evaluation of the repeatability of the replicates (replicate normalization) and the removal of outliers are performed; second, normalization is performed between conditions and the fold change for every gene is estimated. To establish the statistical significance of each fold change, a z test was performed and a P value (P < 0.05) was calculated for each spot ratio using the false discovery rate (FDR) method for false‐positive error correction. Only clones with a two‐fold change or greater, and statistically significant from the z test, were considered for further analysis. The nucleotide sequences of differentially expressed sequence tags contained in the rice macroarray were determined and compared with nucleotide and protein sequence databases (GenBank, http://www.ncbi.nlm.nih.gov; KOME, http://cdna01.dna.affrc.go.jp; Rice Genome Annotation Project, http://rice.plantbiology.msu.edu; MGOS, http://www.mgosdb.org).

Blast resistance assays

Resistance to infection by the rice blast fungus M. oryzae (PR9 isolate) was determined using the detached leaf assay, as described previously (Coca et al., 2004). For this, young leaves of either mycorrhizal or nonmycorrhizal soil‐grown plants were placed into Petri dishes with 1% w/v water—agar containing 2 mg/L kinetin. Whatman filter papers saturated with an M. oryzae PR9 spore suspension at the appropriate concentration were placed onto the upper face of the leaf for 48 h and then removed. The inoculated leaves were maintained in the dark in a chamber under high‐humidity conditions for 48 h and the filter papers were removed. Leaves were maintained at 28 °C and 90% relative humidity under a 16‐h/8‐h light/dark photoperiod for the required period of time. The ability of the fungus to produce spores was estimated by counting the number of spores collected from infected leaves at 7 dpi with the highest spore concentrations (106 spores/mL) (Campos‐Soriano and San Segundo, 2009). Lesion areas were quantified by Image Analysis Software, Assess 2.0, for plant disease quantification (Lamari, 2008). One‐way anovas were used to evaluate differences in the leaf area affected by blast lesions between mycorrhizal and nonmycorrhizal plants. For anovas in which the F test was significant at the probability level of 0.05 or lower, the least‐significant difference test was applied to detect differences.

Total RNA was also obtained from M. oryzae‐infected leaves at different times after inoculation with fungal spores and used for gene expression analysis.

Supporting information

Table S1 Primers used for quantitative reverse transcriptase‐polymerase chain reaction (RT‐qPCR) analysis.

Supporting info item

Click here for additional data file. (47KB, doc)

ACKNOWLEDGEMENTS

This work was supported by the ‘Proyecto Intramural’ 200420E613 from Consejo Superior de Investigaciones Científicas and grant BIO2009‐08719 from the Spanish Ministry of Science and Innovation, as well as by the Consolider‐Ingenio 2010 Programme CSD2007‐00036 ‘Centre for Research in Agrigenomics’. We also thank the ‘Departament d'Innovació, Universitats i Empresa’ from the Generalitat de Catalunya (Xarxa de Referencia en Biotecnología and SGR 09626) for substantial support.

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Associated Data

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Supplementary Materials

Table S1 Primers used for quantitative reverse transcriptase‐polymerase chain reaction (RT‐qPCR) analysis.

Supporting info item

Click here for additional data file. (47KB, doc)

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