The pathogenic white-rot fungus Heterobasidion ...

Tree Physiology 31, 1262–1272 doi:10.1093/treephys/tpr113

Research paper

Jenny Arnerup, Mårten Lind, Åke Olson, Jan Stenlid and Malin Elfstrand1 Department of Forest Mycology and Pathology, Swedish University of Agricultural Sciences, Box 7026, S-750 07 Uppsala, Sweden; 1Corresponding author ([email protected]) Received March 14, 2011; accepted October 5, 2011; handling Editor Torgny Näsholm

Norway spruce [Picea abies (L.) Karst.] is one of the economically most important conifer species in Europe. The major pathogen on Norway spruce is Heterobasidion parviporum (Fr.) Niemelä & Korhonen. To achieve a better understanding of Norway spruce’s defence mechanisms, transcriptional responses in bark to H. parviporum infection were compared with the response WRZRXQGLQJXVLQJF'1$DPSOLÀHGIUDJPHQWOHQJWKSRO\PRUSKLVP7KHPDMRULW\RIWKHUHFRYHUHGWUDQVFULSWGHULYHGIUDJPHQWV (TDFs) showed a similar expression pattern for infection and wounding treatment, although inoculated samples showed an enhanced reaction. Genes related to systemic acquired resistance, e.g., PR1, accumulated after H. parviporum infection. Simultaneously, several transcripts involved in various aspects of jasmonic acid (JA)- and ethylene (ET)-mediated signalling accumulated. Genes involved in the ubiquitin/proteasome system were also regulated. Expression patterns have been FRQÀUPHGE\TXDQWLWDWLYHSRO\PHUDVHFKDLQUHDFWLRQ7KHH[SUHVVLRQSDWWHUQVRIWKHLVRODWHG7')VVXJJHVWWKDWLQIHFWLRQ with H. parviporumLQ1RUZD\VSUXFHLQGXFHVDEURDGGHIHQFHZLWKPDQ\VLPLODULWLHVWRQRQVSHFLÀFGHIHQFHUHVSRQVHVLQ angiosperms. The parallel induction of salicylic acid- and JA/ET-mediated pathways implies spatially separated responses in different cell layers, with and without hyphal contact. A set of TDFs were analysed in an independent experiment with unrelated material treated with wounding or with inoculation with H. parviporum or Phlebiopsis gigantea, verifying the RULJLQDOREVHUYDWLRQVDQGXQGHUOLQLQJWKHQRQVSHFLÀFGHIHQFHUHVSRQVHV,QDGGLWLRQRXUGDWDVXJJHVWWKDWUHURXWLQJRI carbon in secondary metabolism is an integral part of Norway spruce induced defence. We report the sequences of three 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase genes (PaDAHP1, PaDAHP2 and PaDAHP3) and their relative expression in response to wounding and infection with H. parviporum and P. gigantea. The results clearly indicate differential regulation of the three DAHPs in the induced defence responses in Norway spruce. This study gives insights into the central mechanisms in the induced defences in Norway spruce. Keywords: DAHP, gene expression, Heterobasidion parviporum, jasmonic acid, Picea abies, salicylic acid.

,QWURGXFWLRQ Conifers are the most widely distributed gymnosperms in the world (Farjón 1998) and, in Europe, Norway spruce [Picea abies (L.) Karst.] is one of the most important conifer species, both ecologically and economically. The root-rot fungus Heterobasidion annosum sensu lato (s.l.) complex consists of three species in Europe: H. annosum (Fr.) Bref., H. parviporum (Fr.) Niemelä &

Korhonen and H. abietinum (Fr.) Niemelä & Korhonen (Niemelä and Korhonen 1998). This fungus is the most serious Norway spruce pathogen in Scandinavia (Bendz-Hellgren et al. 1998). Heterobasidion annosum s.l. can infect Norway spruce in two ways: (i) when basidiospores colonize fresh wood on a stump or on an injury on the tree and (ii) through root contacts (Asiegbu et al. 2005). In mature Norway spruce trees, the fungus usually

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The pathogenic white-rot fungus Heterobasidion parviporum WULJJHUVQRQVSHFLÀFGHIHQFHUHVSRQVHVLQWKHEDUNRI1RUZD\ spruce

Responses to H. parviporum in bark of Norway spruce

defence responses involving SA are less frequent in conifers (Davis et al. 2002, Piggott et al. 2004, Phillips et al. 2007, Likar and Regvar 2008) and many do not show any effect of methyl salicylate (MeSA) on defence or defence gene regulation (Liu et al. 2003, Piggott et al. 2004). However, the reports indicate a certain extent of antagonistic action to JA (Liu et al. 2003, Phillips et al. 2007). Davis et al. (2002) demonstrated induction of transcripts of chitinases by treatments of JA and SA, while genes involved in secondary metabolism have been shown to be induced by MeJA (Richard et al. 2000, Fäldt et al. 2003). Genes induced by wounding and involved in the biosynthesis of ET have also been characterized (Hudgins et al. 2006, Ralph et al. 2007). The overall picture shows large gaps of knowledge yet to be Àlled regarding conifer defence signalling. Another aspect of induced conifer defence responses that has been studied more thoroughly is the role of secondary metabolites such as phenols, stilbenes, Áavanoids and terpenes in response to pathogen and insect attack (Woodward and Pearce 1988, Lindberg et al. 1992, Ralph et al. 2006). One rate-limiting enzyme for phenolic secondary metabolism is phenylalanine ammonia-lyase (PAL). It is the Àrst enzyme in the phenylpropanoid biosynthesis pathway and is induced upon wounding, insect attack and H. annosum s.l. infections (Ralph et al. 2006, Adomas et al. 2007, Koutaniemi et al. 2007, Likar and Regvar 2008). Molecular studies of the interactions between H. annosum s.l. and its hosts have revealed multiple overlapping defence strategies in Pinaceae, such as expression of defence genes, production of PR proteins and antimicrobial compounds, as well as major shifts in primary and secondary metabolism (Fossdal et al. 2001, 2005, Asiegbu et al. 2003, 2005, Li and Asiegbu 2004, Adomas et al. 2007). However, it has been suggested that these responses are not pathogenspeciÀc (Adomas and Asiegbu 2006). The main objective of this study was to gain basic knowledge of the induced defence responses in Norway spruce to H. parviporum and to compare these responses with the induced wound response. For this purpose we chose the cDNA-ampliÀed fragment length polymorphism (AFLP) method (Bachem et al. 1996) which does not require prior sequence information and has been shown to be a useful screening method for differential gene expression in studies of interaction with pathogens in nonmodel organisms (Durrant et al. 2000, Wang et al. 2010).

0DWHULDOVDQGPHWKRGV Plant and fungal materials, inoculation and RNA preparation Two different spruce materials have been used in this study. For the cDNA-AFLP and quantitative polymerase chain reaction (qPCR) veriÀcation, 10 7-year-old full-sibs were used, which were planted at a research station in Svalöv in southern

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causes stem rot and reduced growth, although normally only seedlings and young trees will die as a result of infection (BendzHellgren and Stenlid 1997). There are no known avirulent strains of H. annosum s.l. and no host genotype in Pinaceae with total resistance (Asiegbu et al. 2005), but experiments with clones of Norway spruce have shown that disease development is partly determined by genetically controlled host tree characteristics (Swedjemark et al. 1997, Swedjemark and Karlsson 2004, Arnerup et al. 2010). Compared with most angiosperm model plants (Arabidopsis thaliana L., Oryza sativa L. and Medicago truncatula Gaertn.), conifers are long-lived organisms that have evolved an efÀcient constitutive chemical and structural defence with a robust barrier defence in the bark (Lindberg et al. 1992, Keeling and Bohlmann 2006). When the barrier defence is broken, induced defence responses are activated. The general knowledge about induced defence responses and their signalling pathways is primarily derived from studies in model plants (primarily A. thaliana). Host reactions depend on the feeding strategy of the pathogen (i.e., biotroph or necrotroph), and different defence pathways are induced and mediated by different hormones (Glazebrook 2005). Salicylic acid (SA) mediates the hypersensitive reaction (HR) and systemic acquired resistance (SAR) in response to biotrophs (Glazebrook 2005). The wound response, also active against necrotrophs, is mediated by jasmonic acid (JA) and ethylene (ET) (Glazebrook 2005). These two major signalling pathways (SA and JA/ET) are essentially antagonistic (Thomma et al. 1998, Kazan and Manners 2008). Yet, recent studies have shown extensive crosstalk between the SA and JA/ET pathways and other hormones, making the picture more complex (Wang et al. 2007, Kazan and Manners 2008, Llorente et al. 2008). Proteomic plasticity is an integral part of plant defence responses. It is intimately linked to major defence signalling pathways and dependent on post-translational modiÀcations and targeted degradation of proteins (Craig et al. 2009). For instance, protein hydrolysis via the ubiquitin/proteasome system (UPS) pathway seems to be a prerequisite for activation of JA-responsive genes. JAZ1 is a repressor of transcriptional activation of JA responses, and when JAZ1 is degraded, transcription of JA response genes is activated (Chini et al. 2007). The current knowledge about induced defence responses in conifers suggests broad similarities to those described in angiosperms. Signalling dependent on JA/ET seems to mediate defence responses to pathogens (Davis et al. 2002, Martin et al. 2002, Hudgins et al. 2006, Ralph et al. 2007). For instance, exogenous application of methyl jasmonate (MeJA) and ET has been shown to induce conifer cellular defence responses (Fäldt et al. 2003, Martin et al. 2002, Hudgins and Franceschi 2004) and to reduce susceptibility to Ceratocystis polonica (Siem.) C. Moreau and Pythium ultimum (Trow.) (Kozlowski et al. 1999, Zeneli et al. 2006). Reports on

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cDNA-AFLP analysis One microgram of total RNA from Àve clones was pooled in each pool, to give two pooled samples of 5 μg total RNA. Poly(A) + RNA was extracted from the pooled sample with the Dynabeads® mRNA PuriÀcation Kit (Invitrogen, Stockholm, Sweden) according to the manufacturer’s instructions. Firststrand cDNA synthesis was performed with RevertAid™ H Minus M-MuLV Reverse Transcriptase and second-strand synthesis was performed essentially as described by Chenchik et al. (1996). All enzymes for the cDNA synthesis were purchased from Fermentas (St. Leon-Rot, Germany). The cDNA was digested with 6 U of EcoRI (NEB) and 4 U of MseI (NEB). To the digested cDNA a ligation mix was added containing 1× T4 reaction buffer, 2.5 pmol of EcoRI adaptor, 25 pmol of MseI adaptor (ABI) and 150 U T4 DNA ligase (NEB). The digestedligated cDNA was diluted 10-fold and used as a template in the preampliÀcation. PreampliÀcation was performed with the EcoRI core primer and MseI core + N (A, T, G, C). Polymerase chain reaction was carried out with the following procedures for the preampliÀcation: denaturation (2 min, 94 °C), then 20 cycles of denaturation

Tree Physiology Volume 31, 2011

(20 s, 94 °C), annealing (30 s, 56 °C) and extension (1 min 72 °C), and a Ànal extension step (15 min, 72 °C). Selective primers used were EcoRI core + NN (GG, GA, GC, GT, AG, AA, AT) and MseI core + NN (AA, AC, AG, AT, CC, CA, CG, CT, GC, GA, GT, TC, TG). Polymerase chain reaction was carried out according to the following touchdown procedures: denaturation (2 min, 94 °C) and then two cycles of denaturation (30 s, 94 °C), annealing (30 s, 65 °C) and extension (1 min 72 °C) followed by 12 cycles where the annealing temperature decreased by 0.8 °C per cycle. The PCR procedure was then completed by 20 cycles of denaturation (30 s, 94 °C), annealing (30 s, 56 °C) and extension (1 min 72 °C) and a Ànal extension step (10 min, 72 °C). Selective PCR products were run on a 6% polyacrylamide gel in a Sequi-Gen® GT Nucleic Acid Electrophoresis Cell (BioRad, Sundbyberg, Sweden) for 2.5 h at 65 W. Gels were silver stained as described by Bassam et al. (1991), rinsed in distilled water and then dried overnight. The intensity of the informative transcript-derived fragments (TDFs) on the polyacrylamide gel was recorded in relation to the untreated control on the day of inoculation. After re-hydration, informative TDFs were extracted from the gel and placed in 10 μl of deionized and ultra Àltered H2O. Bands from the inoculated treatment were only extracted where no expression could be detected in wounding or control. The eluted PCR fragment was diluted 10-fold and used as a template for re-ampliÀcation. Re-ampliÀed fragments were separated on 1.8% agarose gel. Polymerase chain reaction products with sharp and clear bands were puriÀed with the AMPURE PCR cleanup kit and sent to Macrogen (Seoul, Korea) for direct sequencing with the EcoRI core primer. Weak and doublebanded products were Àrst cloned with the TOPO-TA cloning kit (Invitrogen) according to the manufacturer’s recommendations, and then sequenced. The quality of the sequences was evaluated manually, and in most cases it was possible to identify the MseI primer sequence. To identify the corresponding gene and to assign a functional annotation to the TDFs, a database search was performed using the BLAST Network Service with the BLASTx algorithm (NCBI nucleotide collection database, National Center for Biotechnology Service; http://www.ncbi.nlm.nih.gov/BLAST) (E-value cutoff = 1e−4). For the TDFs where no corresponding annotated gene for a conifer was found, a BLASTx search was performed using The Arabidopsis Information Resource; http:// www.arabidopsis.org/index.jsp (E-value cutoff = 1e−4). For some TDFs, a BLASTn search using the NCBI nucleotide collection database was performed (E-value cutoff = 1e−20). The retrieved cDNA Picea spp. sequences were further analysed using BLASTx. The TDFs were placed in functional categories according to their Gene Ontology (GO) annotations from the TAIR database. All TDFs in Supplementary Table S1 available as Supplementary Data at Tree Physiology Online have been submitted to the dbEST database at NCBI under accession numbers HS032497–HS032614 and HS032615–HS032617.


Sweden. Branches were artiÀcially inoculated with a strain of H. parviporum (Rb175) originally (Stenlid 1987). Inoculations were also made in an unrelated material of 2-year-old Norway spruce plants from a plant nursery. The trees were planted in pots, kept in a greenhouse, and inoculated with H. parviporum (Rb175) or Phlebiopsis gigantea [Fr.] Jülich (RotstopS). The nursery material was used for qPCR analysis and three plants were used as biological replicates for each treatment. To treat the plants, a 5 mm circular wound was made with a cork borer. Wooden plugs infected with H. parviporum or P. gigantea were attached to the circular wound and both inoculated and un-inoculated control wounds were sealed with ParaÀlm®. For the full-sib material, inoculation was made on branches from the previous year’s growth. On each branch, three wounds were made and pooled as one sample. Samples were harvested at 3, 7 and 14 days post inoculation (dpi). Unharmed controls, i.e., bark not previously wounded, were collected at the time of inoculation and at 14 dpi. At harvest, bark surrounding the wound was cut out with the help of a 10 mm cork borer to standardize the size of the bark sample. For the unrelated material, the inoculation was performed as described above, but on the stem ~10 cm from soil level, and samples were harvested at 5 dpi. In addition to the samples harvested at the point of inoculation, samples were also taken at a distance of 5 cm above the point of inoculation on the nursery plants. The samples were immediately frozen in liquid nitrogen and stored at –80 °C until use. Total RNA was isolated as described by Chang et al. (1993). To eliminate contamination by genomic DNA, the total RNA was treated with DNaseI (Sigma-Aldrich, Stockholm, Sweden) before use.


Quantitative PCR

Quantitative PCR analysis was carried out with the software tool REST (PfafÁ et al. 2002). The mathematical model in REST used to calculate the relative expression ratio is (E target )ΔC1target(control− sample) /(Eref )ΔC1ref(control − sample). The real-time PCR efÀciency was calculated from the slope of the standard curve, according to the established equation E = 10[ −1/ slope] Changes in relative transcript abundance between treated trees (wounded vs. inoculated) and between treated trees and untreated control trees were compared using a pairwise reallocation test with 10,000 permutations (PfafÁ et al. 2002).

Results ,GHQWLÀFDWLRQRIGLIIHUHQWLDOO\H[SUHVVHGJHQHV by cDNA-AFLP To identify processes associated with defence against H. parviporum, transcriptional responses in Norway spruce bark inoculated with H. parviporum have been compared with the responses to wounding using cDNA-AFLP analysis. Bark samples were harvested at 3, 7 and 14 dpi and unharmed bark was used as negative control. cDNA-AFLP patterns generated with 54 different primer combinations allowed a screening of ~2500 TDFs on polyacrylamide gels ranging from 50 to 1200 bp in size. On average, 10.5 fragments per primer combination showed a pattern with differential band intensity between treatments and control. One hundred and ninety-nine TDFs were chosen for further investigations based on intensity of the band of inoculated treatment in relation to either unharmed bark control or wounding treatment. To avoid false– positives, the experiments were carried out with two biological replicates. Of the sequenced TDFs, 176 gave a reliable sequence, and several TDFs were sequenced multiple times with different primer combinations. Depending on the primers, the ampliÀcation efÀciency varied, which gave rise to an intensity difference between the bands on the polyacryamide gel. However, the relation between treatments agreed. In total, 149 TDFs were represented by one single sequence; including 30 TDFs with no signiÀcant hit to the databases (NCBI or TAIR) using either BLASTn or BLASTx, while 11 TDFs showed similarities to conserved proteins without known function. In this system the TDFs could potentially originate from both spruce and H. parviporum. All TDFs with no signiÀcant hit to a gymnosperm sequence using BLASTn (NCBI) or BLASTx (TAIR) were analysed with a BLASTn search in the fully sequenced H. annosum s.l. genome (Å. Olson et al., manuscript in preparation). In total, 10 TDFs with a signiÀcant hit to H. annosum s.l. were found (data not shown). The putative homology, accession numbers and GO category assignment for 119 TDFs are shown in Supplementary Material 1A available as Supplementary Data at Tree Physiology Online (70 up-regulated) and Supplementary Material 1B available as Supplementary Data at



For isolation of full-length cDNA sequences of Norway spruce DAHP genes, the SMARTer RACE cDNA ampliÀcation kit (Clontech, Saint-Germain-en-Laye, France) was used according to the manufacturer’s instructions to perform rapid ampliÀcation of cDNA ends (RACE), RT reactions and 5′/3′ RACE reactions. Primer sequences can be found in Supplementary Table S2 available as Supplementary Data at Tree Physiology Online. The isolated FLcDNAs are deposited in GenBank (GenBank: HQ441161, HQ441162 and HQ441163). To verify the banding pattern obtained from the cDNA-AFLP analysis, qPCR was carried out on a selection of differentially expressed TDFs (see Supplementary Material 1 available as Supplementary Data at Tree Physiology Online). The expression patterns of the TDFs were further veriÀed in the nursery material. Each treatment and time point was represented by three biological and three technical replicates. Primers and probes were designed from the ampliÀed TDF sequences using the Primer3 software, with a melting temperature (Tm) between 58 and 60 °C, and produced amplicons between 82 and 142 base pairs (bp) (Supplementary Material 2 available as Supplementary Data at Tree Physiology Online). Primer speciÀcity was veriÀed on agarose gel and by melt-curve analysis and the PCR efÀciency was between 93 and 105%. The relative expression of two additional isoforms of the 3-deoxy-darabino-heptulosonate 7-phosphate synthase genes (DAHP) has also been examined by qPCR. Primers were designed based on sequences from Picea glauca (GenBank: BT118499, BT115938 and BT114027) (Supplementary Material 2 available as Supplementary Data at Tree Physiology Online). Total RNA (1 μg) was reverse transcribed with the iScript™ cDNA synthesis kit (Bio-Rad, Sundbyberg, Sweden). Prior to reverse transcription, total RNA was treated with DNase1 (Sigma-Aldrich). The cDNA synthesis was diluted 1:1 in deionized water, and 1 μl was used as a template in the qPCR reaction. The qPCR was performed on the iQ™5 Multicolor Real-Time PCR Detection System (Bio-Rad) and the thermalcycling condition parameters were as follows: 95 °C for 10 min, 40 cycles of 95 °C for 15 s, 60 °C for 1 min. A linear plasmid standard curve was used to measure the PCR efÀciency. Multiplexed reactions with TaqMan probes labelled with HEX or FAM, paired with a BHQ, as well as SYBR-Green reactions were conducted using the Fermentas Maxima® Probe/ ROX qPCR Master Mix kit and the Maxima® SYBR Green/ Fluorescein qPCR Master Mix kit. Multiplexing parameters were as stated in Supplementary Material 2 available as Supplementary Data at Tree Physiology Online. Transcript abundance was normalized to the constitutive expressed genes α-Tubulin (αTUB) and Elongation factor 1-α (ELFα), which both showed low variation between samples. Elongation factor 1-α has previously been documented as a good gene for normalization (Bedon et al. 2007).

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1266 Arnerup et al. Tree Physiology Online (49 down-regulated). Around 20% each of the up-regulated TDFs were assigned to GO categories associated with biotic stress or to GO categories with unknown molecular functions. Among the down-regulated TDFs, the largest individual GO category was ‘biological process unknown’ (35% of the TDFs).

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Analysis of TDFs associated with hormone signalling pathways Genes involved in defence signalling were selected for testing in a genetically unrelated material of nursery-grown plants. The plants were subjected to three treatments: wounding, inoculation with H. parviporum or inoculation with P. gigantea (a nonpathogen on conifers). Material was harvested at the point of inoculation and 5 cm above the inoculation site at 5 dpi. The pattern of up- and down-regulation at the site of inoculation in this material was very similar to the pattern seen in the experiment with the full-sib material. LOX, ACO, ACS and LURP1 showed a strong up-regulation after H. parviporum and P. gigantea inoculation compared with wounding. PR1 was highly induced in all treatments. PIN1 was more down-regulated in H. parviporum and P. gigantea inoculated samples (Table 1). No signiÀcant difference was observed for the expression patterns between samples inoculated with H. parviporum and P. gigantea. In the distal samples ACO showed a differential regulation between H. parviporum and wounding while the other genes were similarly expressed in both treatments (Table 1).

([SUHVVLRQRI'$+3 DAHP is the Àrst enzyme in the shikimate pathway and it controls carbon Áow from carbohydrate metabolism to the biosynthesis of aromatic compounds and many secondary metabolites (Herrmann 1995, Herrmann and Weaver 1999). The observed down-regulation of DAHP (TDF JA09_504) was contradictory to previous observations in conifers (Ralph et al. 2006,


Discussion The main goal of this study was to identify transcriptional changes associated with induced defence responses in Norway spruce to the necrotrophic fungus H. parviporum. These changes were compared with induced wound responses and with induced responses to the saprotrophic fungus P. gigantea. Previous studies of the H. annosum s.l.–conifer pathosystem suggest that differences between trees in susceptibility to H. annosum s.l. are a quantitative trait (Swedjemark et al. 1997, Arnerup et al. 2010), and it has been argued that conifer defence responses towards H. annosum s.l. may be more organ-speciÀc than pathogen-speciÀc, at least in young seedlings (Adomas et al. 2007). The overall results from our study are in agreement with the suggestion that the defence response is not speciÀc to H. annosum s.l. infection, as our results convey the picture that the response in Norway spruce to H. parviporum is a broad, non-speciÀc defence response with large similarities to the wound response. The cDNA-AFLP method is an adequate method for screening gene expression in non-model organisms. As the method relies on separation of unknown cDNA fragments by size, it is important to keep in mind that each band from the AFLP reac-


To validate the cDNA-AFLP data, the expression patterns of 18 TDFs, corresponding to ~11% of the data set, were examined by qPCR (see Supplementary Material 2 available as Supplementary Data at Tree Physiology Online). Transcript-derived fragments from inoculated and wounded samples were selected based on their putative function in defence, and their expression was compared with the expression in unharmed bark. Overall, corresponding results (up- and down-regulation) were obtained for all transcripts between the two techniques except for MLO11 (Supplementary Material 1 available as Supplementary Data at Tree Physiology Online, Figure 1). The qPCR veriÀed the difference in magnitude of the regulation between H. parviporum inoculation and wounding treatment for most genes (Supplementary Material 1 available as Supplementary Data at Tree Physiology Online, Figure 1).

Adomas et al. 2007). To investigate this further, three full-length cDNAs corresponding to PaDAHP1, PaDAHP2 and PaDAHP3 (GenBank: HQ441161, HQ441162 and HQ441163) were isolated by RACE. BLASTx analysis identiÀed corresponding orthologues for PaDAHP1, PaDAHP2 and PaDAHP3 in P. glauca (GenBank: BT118499, BT115938 and BT114027) and Pinus spp. (TC133985, TC117608 and TC118521), and for PaDAHP1 and PaDAHP2 in Picea sitchensis (GenBank: EF677076 and EF084957). Primers designed for the three isolated DAHP gene sequences were used to determine the relative expression levels of the genes by qPCR (see Supplementary Material 1 available as Supplementary Data at Tree Physiology Online) in the full-sib material and in the unrelated nursery material (Figure 2a and b, respectively). PaDAHP1 was signiÀcantly down-regulated at all time points in both treatments in the fullsib material (P < 0.05, Figure 2a). There was also a signiÀcant difference between inoculated and wounded samples in the full-sib material at 7 and 14 dpi for PaDAHP1 (P < 0.05, Figure 2a). No signiÀcant change was observed in PaDAHP1 expression in the nursery material (Figure 2b). An up-regulation of PaDAHP2 was detected in both materials (Figure 2a and b), which was signiÀcant at all time points after H. parviporum inoculation on the full-sib material (P < 0.05, Figure 2a). The up-regulation was signiÀcantly higher compared with wounded material at 7 and 14 dpi (P < 0.05, Figure 2a). Compared with unharmed control material, the expression of PaDAHP3 was down-regulated in all treatments (Figure 2a and b).


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Figure 1. Quantitative PCR analyses of wounded or H. parviporum inoculated bark samples of 7-year-old full-sib spruce plants. Steady-state RNA levels were determined by qPCR with gene-speciÀc primers. Samples were harvested at 3, 7 and 14 dpi. Three or four biological replicates were used and all data have been normalized to the constitutive expressed genes Elongation-factor-1α and α-Tubulin. Treatments are as follows: wounding (open bars) and inoculation with H. parviporum (grey bars). The error bars correspond to the standard deviation. Asterisks indicate if the expression is signiÀcantly different from the unharmed control ( P < 0.05,

P < 0.01,

P < 0.001). Lines over bars indicate that there was no signiÀcant difference between the wounding and inoculation treatments.


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Table 1. Expression patterns of selected TDFs in the nursery material. Samples harvested at 5 dpi at the point of inoculation (0) and at a distance of 5 cm from the point of inoculation (5). Hp, H. parviporum inoculated; W, wounded; Pl, P. gigantea inoculated. Hp_0

JA09_315/ACO JA09_134-2/ACS JA09_465/LOX JA09_528/PR1 JA09_395/PIN1 JA09_34/LURP JA09_381/CYP450 JA09_241/AGD11 JA09_244/Zinc Ànger

W_0

Pg_0

Hp_5

W_5

E. ratio1

SE2

E. ratio

SE

E. ratio

SE3

E. ratio

SE

E. ratio

SE

7.0 7.3 5.8 17.7 −6.3 2.3 7.6 1.3 1.05

3.1 4.5 3.1 1.1

3.4 0.4

5.4 4.7 1.4

2.5 1.4 3.2 15.8 −3.5 0.2 7.5 −0.1 −0.3

1.7

1.6 2.0 3.6 1.1 0.3 1.1 0.2 0.6

9.3 3.0 8.0 18.5 −6.9 2.4 9.0 -

0.7 5.7 1.7 0.8 1.1 2.3 1.8

1.7 3.0 1.0 1.0 −3.3 −4.2 1.5 -

1.8 2.4 3.4 1.1 2.0

0.9

2.0

−6.8 2.2 3.1 0.9 −2.9 −4.7 2.0 -

0.8

3.1 1.9 0.7 0.8 0.8

1.2

Figure 2. Expression pattern of three DAHP genes. Three biological replicates were used, and all data have been normalized to the constitutively expressed genes Elongation-factor-1α and α-Tubulin. Samples were harvested at 3, 7 and 14 dpi for the full-sib material and at 5 dpi for the nursery material. Treatments are as follows: wounding (open bars) and inoculation with H. parviporum (striped bars) or P. gigantea (cross-hatched). The error bars correspond to standard deviation. Asterisks indicate if the expression is signiÀcantly different from the unharmed control (

P < 0.001,

P < 0.01, P < 0.05). Lines over bars indicate that there was no signiÀcant difference between the wounding and inoculation treatments. (a) Expression patterns in the full-sib material at 3, 7 and 14 dpi. (b) Expression patterns in the nursery material at 5 dpi.

tion may comprise more than one cDNA fragment. For this reason it is essential to verify the expression pattern with a technique such as qPCR or northern blotting. We have veriÀed the expression of ~11% of the TDFs presented in this study. All TDFs tested have a documented function in different aspects of plant defence (Figure 1). Central defence pathways are conserved among angiosperms (Baumberger et al. 2003, Tsai et al. 2006, Paterson et al. 2010) and probably also in gymnosperms (Ralph et al. 2006, Likar and Regvar 2008) but the defence signalling pathways are comparatively much less explored in conifers. There are relatively few functionally characterized proteins in conifers compared with angiosperms like Arabidopsis and no conifer genome has so far been sequenced. The annotation of the TDFs has therefore been performed against Arabidopsis, which is the best characterized plant genome. As a result of the length of some TDFs (ranging from 100 to ~800 bp) and the fact that the BLAST analysis is done against a relatively distant species, the statistical conÀdence of the BLAST analyses will


vary. In addition, there is a relatively large number of TDFs with no signiÀcant hit to any known gene (~15%). A higher proportion of TDFs without homology can be expected in interaction studies, as conifers activate specialized functions when confronted with pathogens or pests (Ralph et al. 2008). A central part of conifer defence responses against wounding and invading pathogens and pests is the production of secondary metabolites, such as mono- and di-terpenes as well as many phenolic compounds (Keeling and Bohlmann 2006). Based on previous studies, we expected to see up-regulation of genes encoding terpene synthases (TPS) (Martin et al. 2002, Hudgins and Franceschi 2004, Phillips et al. 2007). Only two TDFs with similarity to TPS genes were recovered, both of which were slightly down-regulated at the end of the experiment (Supplementary Material 1 available as Supplementary Data at Tree Physiology Online). The absence of up-regulated TPS genes is probably due to the fact that the samples analysed contained little to no xylem tissue as the largest induction of TPS genes is normally found in the developing xylem (Martin et al. 2002).


1Expression ratio of treated samples compared with unharmed bark samples, Log2. Bark samples were harvested from three independent treated plants for each treatment. All data have been normalized to the constitutive expressed genes Elongation-factor-α and α-Tubulin. 2Standard error. Asterisks indicate if the expression is signiÀcantly different from the unharmed control ( P < 0.05,

P < 0.01,

P < 0.001). 3Only two biological replicates.


considered to be a marker for SAR in plants (Thomma et al. 2001), while LURP1 is the most active member of a cluster of genes co-expressed with PR1 during SAR, known as the PR1 regulon in Arabidopsis (Knoth and Eulgem 2008). Transcripts of PR1 were accumulated concomitantly with a TDF with similarity to LURP1, indicating accumulation of SA in inoculated tissues. It is known that inoculation with H. annosum can lead to accumulation of SA in young Norway spruce seedlings (Likar and Regvar 2008). Histochemical studies in spruce inoculated with H. annosum have shown that HR and necrosis seem to occur in the cell layers closest to the wound (Solla et al. 2002, Johansson et al. 2004). Exogenous application of MeJA, ET or MeSA does not induce characteristic tissue damage associated with HR (Hudgins and Franceschi 2004), indicating the necessity of other signalling agents. In this context, it is worth noticing that P. gigantea inoculation induces a similar accumulation of PR1 and LURP1 transcripts as H. parviporum inoculation, yet the strong induction of PR1 (nearly 20 times the control) suggests an elevated SA level in Norway spruce tissues. This raises the question of where the accumulation of SA would be found in relation to the invading hyphae and in relation to the biosynthesis of JA and ET. In Arabidopsis, SA- and JA/ET-mediated pathways are considered to be antagonistic and induced differently, depending on the feeding strategy of the pathogen (Glazebrook 2005). The responses we see can be either a temporal and spatial co-activation of different signalling pathways, or responses spatially separated to different cells depending on the existence of fungal hyphae in the tissue. Essentially, we found a wound response with low induction of JA/ET biosynthesis genes in samples distal to the inoculated zone, suggesting that most of the transcriptional activity of the TDFs we have isolated occurs in the vicinity of the H. parviporum mycelium. Most likely the separation of responses can be found between the cell layers in contact with the hyphal front. Recent studies have shown that there is extensive crosstalk between the SA and JA/ET pathways as with other phytohormones such as auxin (Wang et al. 2007, Llorente et al. 2008). In addition to activation of the major defence signalling pathways, we observed possible signs of crosstalk between signalling pathways; for instance, a TDF representing PaPIN1 was strongly repressed after inoculation and wounding. The repression of PaPIN1 may indicate a reduced amount of free auxin, since the expression of this gene has been shown to be correlated with an accumulation of polar auxin in Norway spruce embryos (Palovaara et al. 2010). In addition, a TDF with similarity to ARF6 was down-regulated in our material. A connection between JA and auxin signalling involving ARF-mediated induction of JAZ1 has been demonstrated by Grunewald et al. (2009). The most likely explanation to our observations is the possible existence of crosstalk and complex signalling



We recovered TDFs with similarity to PAL and transcinnamate 4-hydroxylase (C4H), both found at the entry point of the phenylpropanoid pathway. These TDFs showed an expected up-regulation in both wounded and inoculated treatments. There are several reports of up-regulation of genes in the phenylpropanoid pathway after infection with H. annosum (Adomas et al. 2007, Koutaniemi et al. 2007, Likar and Regvar 2008). Koutaniemi et al. (2007) report a strong induction of PaPAL2 and PaC3H2 in response to H. parviporum infection. In young Pinus sylvestris roots, infection with H. annosum alters the expression of three C4H transcripts (Adomas et al. 2007). These observations indicate that changes in the preferred isoforms of enzymes in the phenylpropanoid pathway may occur in defence, resulting in changes in end products. Our results indicate a change of the preferred isoform of DAHP. DAHP catalyses the Àrst step in the shikimic acid pathway (Herrmann 1995) and is co-induced with PAL which catalyses the Àrst step in the phenylpropanoid pathway (Logemann et al. 2000). The veriÀed down-regulation after inoculation with H. parviporum of the TDF similar to DAHP, PaDAHP1 (Supplementary Material 1 available as Supplementary Data at Tree Physiology Online, Figure 2), was therefore unexpected. PaDAHP1 shows the highest constitutive expression in Norway spruce. However, after H. parviporum inoculation or wounding, a signiÀcant shift in transcript abundance was observed where PaDAHP1 was down-regulated and PaDAHP2 up-regulated. This is in agreement with previous studies, where transcripts homologous to PaDAHP2 have been shown to be up-regulated in conifers in response to wounding (Ralph et al. 2006) and inoculation with H. annosum (Adomas et al. 2007). The shift in DAHP expression may be important for enhanced carbon Áow from protein synthesis to secondary metabolism. In beech (Fagus sylvatica), transcript accumulation of DAHP and downstream genes in the shikimic acid pathway after ozone treatment is associated with an increase in cell wallbound phenolics and Klason lignin (Betz et al. 2009). The two major induced defence pathways in plants are considered to be mediated by SA and JA/ET. In our work we see simultaneous induction of SA- and JA/ET-mediated pathways after inoculation of Norway spruce with H. parviporum. As LOX is induced in inoculated material, it is likely that the octanodecanoid pathway and production of JA are activated. Furthermore, the up-regulation of ACS and ACO, together with a methionine synthase (JA09_300, Supplementary Material 1 available as Supplementary Data at Tree Physiology Online), suggests that ET production is induced in inoculated tissues. We see an up-regulation of a TDF with similarity to ATERF-1, which is a positive regulator of JA/ET-mediated defence genes (Lorenzo et al. 2003). There is also an up-regulation of JAZ1, which is a repressor of JA-mediated transcription (Chini et al. 2007), possibly to replenish JAZ1 in the cells. The PR1 gene is

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6XSSOHPHQWDU\GDWD Supplementary data for this article are available at Tree Physiology Online.

$FNQRZOHGJPHQWV The authors thank Dr Heriberto Vélëz and Dr Elizabeth Bent for critically reading the manuscript.


)XQGLQJ This study was supported by grants from the Knowledge Foundation, the Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning, and the Swedish Foundation for Strategic Research. The Ànancial support was also provided by the Research School in Forest Genetics and Breeding at the Swedish University of Agricultural Science (SLU).

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networks between phytohormones in Norway spruce, reminiscent of those in Arabidopsis. The UPS is essential for most aspects of cellular regulation and several aspects of signalling and regulation of biotic stress responses (Dreher and Callis 2007, Craig et al. 2009). E3 ubiquitin ligases are responsible for the Ànal tagging of proteins, thereby conferring speciÀcity to the degradation process. We isolated three TDFs up-regulated in inoculation treatments with similarity to PUB23, ATL6 and Xerico E3-ubiquitin ligases, all of which respond to MAMPs in Arabidopsis (Libault et al. 2007). In Arabidopsis, the PUB22/PUB23/PUB24 triplet acts as a negative regulator of plant defence responses (Yee and Goring 2009), and it is highly induced after treatment with the MAMPs Ág22, chitin and elf18 (Serrano et al. 2006, Libault et al. 2007, Trujillo et al. 2008). The triple mutant pub22/ pub23/pub24 shows an enhanced oxidative burst and enhanced resistance to oomycete and bacterial pathogens, thus indicating that PUB22/PUB23/PUB24 are negative regulators of the oxidative burst (Trujillo et al. 2008). The recovery of a gene putatively involved in limiting the oxidative burst in a necrotrophic pathosystem is thought provoking, as the oxidative burst generally occurs during a hypersensitive response that confers resistance to biotrophic pathogens (Glazebrook 2005). The invasion of a necrotrophic pathogen such as H. parviporum can actually be assisted by the cellular damages caused by an oxidative burst and the subsequent HR (Govrin and Levine 2000). This hypothesis may be supported by the observed down-regulation of HIN1, previously reported to be associated with HR (Gopalan et al. 1996). In conclusion, we used the cDNA-AFLP approach to screen for genes differently expressed in H. parviporum inoculated compared with wounded Norway spruce bark. A number of induced genes were identiÀed, suggesting a broad induced defence with many similarities to non-speciÀc defence responses in angiosperms. A strong induction of the SAR marker-gene PR1 was recorded at the same time as several transcripts involved in various aspects of JA-mediated regulation, corresponding to a spatially separated response to different cell layers with and without hyphal contact. Moreover, our data suggest that rerouting of carbon in the secondary metabolism is an integral part of the Norway spruce induced defence.


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