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JXB Advance Access first published online on February 3, 2008
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Journal of Experimental Botany, doi:10.1093/jxb/erm317
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© 2008 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)

RESEARCH PAPER

Peach [Prunus persica (L.) Batsch] KNOPE1, a class 1 KNOX orthologue to Arabidopsis BREVIPEDICELLUS/KNAT1, is misexpressed during hyperplasia of leaf curl disease

Giulio Testone1 *, Leonardo Bruno2 *, Emiliano Condello1, Adriana Chiappetta2, Alessandro Bruno2, Giovanni Mele1, Andrea Tartarini3, Laura Spanò3, Anna Maria Innocenti2, Domenico Mariotti1, Maria Beatrice Bitonti2 and Donato Giannino1,{dagger}

1Institute of Biology and Agricultural Biotechnology, National Research Council of Italy (CNR), via Salaria km 29,300, 00015 Monterotondo Scalo, Rome, Italy
2Department of Ecology, University of Calabria, Ponte Bucci, 87030 Arcavacata di Rende, Cosenza, Italy
3Department of Basic and Applied Biology, University of L'Aquila, Via Vetoio, Coppito 67010, L'Aquila, Italy

{dagger} To whom correspondence should be addressed. E-mail: donato.giannino{at}ibba.cnr.it

Received 19 October 2007; Revised 20 November 2007 Accepted 22 November 2007


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Supplementary data
 References
 
Class 1 KNOTTED-like (KNOX) transcription factors control cell meristematic identity. An investigation was carried out to determine whether they maintain this function in peach plants and might act in leaf curliness caused by the ascomycete Taphrina deformans. KNOPE1 function was assessed by overexpression in Arabidopsis and by yeast two-hybrid assays with Arabidopsis BELL proteins. Subsequently, KNOPE1 mRNA and zeatin localization was monitored during leaf curl disease. KNOPE1 and Arabidopsis BREVIPEDICELLUS (BP) proteins fell into the same phyletic group and recognized the same BELL factors. 35S:KNOPE1 Arabidopsis lines exhibited altered traits resembling those of BP-overexpressing lines. In peach shoot apical meristem, KNOPE1 was expressed in the peripheral and central zones but not in leaf primordia, identically to the BP expression pattern. These results strongly suggest that KNOPE1 must be down-regulated for leaf initiation and that it can control cell meristem identity equally as well as all class 1 KNOX genes. Leaves attacked by T. deformans share histological alterations with class 1 KNOX-overexpressing leaves, including cell proliferation and loss of cell differentiation. Both KNOPE1 and a cytokinin synthesis ISOPENTENYLTRANSFERASE gene were found to be up-regulated in infected curled leaves. At early disease stages, KNOPE1 was uniquely triggered in the palisade cells interacting with subepidermal mycelium, while zeatin vascular localization was unaltered compared with healthy leaves. Subsequently, when mycelium colonization and asci development occurred, both KNOPE1 and zeatin signals were scattered in sectors of cell disorders. These results suggest that KNOPE1 misexpression and de novo zeatin synthesis of host origin might participate in hyperplasia of leaf curl disease.

Key words: KNOPE1, peach, role in cell undetermined fate, response in leaf curl disease, Taphrina deformans, transcription factor, zeatin


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Supplementary data
 References
 
The plant KNOTTED1-like (KNOX) genes encode homeodomain- (HD) containing transcription factors (TFs) and fall into two classes, distinguished by the identity grade of the residues within the HD, intron position, and expression patterns (Reiser et al., 2000; Hake et al., 2004).

Class 1 KNOX genes are differentially required for shoot apical meristem (SAM) establishment and function, and constitute a pathway that controls meristem cell fate (Hake et al., 2004). In simple leafed species, they are typically expressed in the SAM, but the down-regulation is required both in cell groups (P0) that will originate a leaf primordium and throughout leaf development (Hay and Tsiantis, 2006).

The Arabidopsis SHOOT MERISTEMLESS (STM) gene suppresses differentiation throughout the SAM, allowing stem cell amplification before cell recruitment into lateral organs (Scofield and Murray, 2006). Arabidopsis KNAT1, also named BREVIPEDICELLUS (BP), KNAT2, and KNAT6 are active in the SAM, but show different spatial patterns of expression compared with that of STM (Reiser et al., 2000). Mutants deficient in these genes do not exhibit alterations in SAM development or function (see references in Scofield and Murray, 2006), suggesting that STM is critical for SAM development. Genetic studies indicate that each class 1 KNOX gene may control different developmental pathways (Hake et al., 2004), although functional redundancy between STM and BP was assessed (Byrne et al., 2002).

In Arabidopsis SAM, BP mRNA is excluded from P0, and marks the peripheral zone (PZ), rib zone (RZ), and the boundaries at the base of leaf primordia. It localizes in the stem below the SAM and in the cells surrounding stem veins (Lincoln et al., 1994). BP is also active in the inflorescence meristem (but not in the floral one), gynoecium, style, and cortical tissues of pedicels (Lincoln et al., 1994; Douglas et al., 2002; Douglas and Riggs, 2005). BP loss-of-function mutants (bp) exhibit growth reduction of tissues that normally express BP, except for the carpel tissues (Douglas et al., 2002; Venglat et al., 2002). The morphological alterations (e.g. shortened internodes and pedicels, enhanced lignin deposition, epinasty of siliques) are due to reduced cell division and impaired differentiation in the affected tissues. Transcript profiling analyses suggest that BP also regulates some lignin pathway genes during internode development (Mele et al., 2003). BP ectopic expression causes the formation of abnormal leaves with intense lobing at margins and, sporadically, the development of ectopic shoot meristems on the leaf adaxial surface (Chuck et al., 1996; Ori et al., 2000; Frugis et al., 2001). Histological analyses evidence the inhibition of proper leaf cell differentiation (e.g. palisade layer and spongy mesophyll cells are hardly distinguishable), which is proposed not to be caused by direct promotion of cell division by KNOX (Scofield and Murray 2006), although cell proliferation was specifically observed during lobe development in STM-overexpressing leaves (Lenhard et al., 2002).

Phytohormones and class 1 KNOX cooperate to regulate the balance between SAM maintenance and the production of lateral organs (Hay et al., 2004). In the Arabidopsis SAM, STM inhibits ASYMMETRIC LEAVES (AS) gene expression, allowing KNAT1/2/6 activity. Class 1 KNOX repress gibberellin synthesis and show reciprocal up-regulation with cytokinins (CKs). In leaf primordia, the auxin and AS cooperate to repress BP (Hay et al., 2006). Moreover, de novo CK biosynthesis, due to ISOPENTENYLTRANSFERASES (IPT) up-regulation, was observed in both Arabidopsis and rice leaves overexpressing STM (Jasinski et al., 2005; Yanai et al., 2005) and STM/BP orthologues, respectively (Sakamoto et al., 2006). Conversely, bacterial IPT overexpression induced KNAT1 and STM ectopic activity (Rupp et al., 1999).

KNOX and BELL proteins form transcriptional complexes, necessary for normal KNOX function in the SAM (Bellaoui et al., 2001). The interactions are selective because each KNOX recognizes a subset of BELL. BP specifically binds to PNY and PNF, encoded by the PENNYWISE (PNY) and POUND-FOOLISH (PNF) genes, which contribute to specify inflorescence internode patterning and floral primordia (Smith and Hake, 2003; Smith et al., 2004).

Clearly, KNOX have been widely investigated in model species, but much less in commercially important trees. KNOX may govern traits of agronomical interest (e.g. for BP orthologues: caulis development, tree habitus, etc.) and there is a growing focus on the roles of KNOX in stem secondary growth of forest trees (Groover et al., 2006). Studies on fruit tree KNOX have been limited to gene characterization (Watillon et al., 1997) and bud expression (Brunel et al., 2002) in apple trees, and to gene function in palm leaf (Jouannic et al., 2007). Here, evidence is provided that peach KNOPE1 shared equivalence at the expression, functional, and biochemical levels with the class 1 BP.

There is a body of evidence that TFs participate in biotic stress responses (Eulgem, 2005), though little is known about the roles of KNOX. Transcriptome analyses revealed root KNAT6 down-regulation after nematode infections (Füller et al., 2007), while Medicago truncatula root KNOX genes were triggered by rhizobia and nematodes (Koltai et al., 2001). Taphrina deformans (Berk.) Tul. is a biotrophic parasitic fungus that causes leaf curl of peach, a disease that seriously affects both crop yield and tree longevity (Rossi et al., 2006, 2007). Histological analyses of expanded leaves infected by T. deformans showed that differentiated palisade cells resumed division capacity, generating hyperopic sectors characterized by undistinguishable palisade and spongy mesophyll layers (Syrop, 1975a). This response was accompanied by the increase of CK content (Sziràki et al., 1975). These features strongly resemble the alterations of BP-overexpressing leaves (Chuck et al., 1996; Frugis et al., 2001). Hence, experiments were conducted to verify whether KNOPE1 transcription occurred during leaf hyperplasia, and KNOPE1 re-activation was observed, together with host zeatin accumulation, at distinct disease stages, suggesting a role for KNOPE1 in the histological disorders.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Supplementary data
 References
 
Plant materials and growth conditions
The IBBA-CNR field-hosted 40 adult peach plants spaced at a distance of 5 mx5 m and pruned to an open-centre shape (vase). These belonged to two clonal lines (S1 and S2) obtained by micropropagation (Giannino et al., 2000, 2003) of two seedlings from the mother plant OP16 (open pollinator 16, Prunus persica cultivar ‘Chiripa’). The level of homozygosity of ‘Chiripa’ was not assessed and it was assumed that it was on average 65%, as estimated by microsatellite fingerprinting (Aranzana et al., 2003). To ensure a grade of plant material homogeneity, only the outdoor adult clones within the S1 line were used for leaf curl disease experiments. S1 clones showed uniform phenotypical and physiological traits (e.g. blooming time, fruit production); however, they may contain a grade of in vitro induced somaclonal variation, which was not assessed.

Plant 18 from the S1 line (F0S1–18) was self-pollinated under controlled conditions, by covering flowers with paper bags in the first 2 weeks of March. Fruit were harvested (July) and seeds were sun dried, treated with Caffaro powder (2 g for 1 kg of seeds) containing 16% copper oxiclorure (ISAGRO, Milan, Italy), stored in paper bags at 7 °C, sown after 90 d, and grown in the greenhouse at 22–25 °C, 16/8 h of light/dark with a light intensity of 100 µmol m–2 s–1 of photosynthetically active radiation (PAR). The seeds generated F1S1–18 individuals, which provided samples at different developmental stages for gene cloning, Southern blot, tissue-specific expression, in situ hybridizations in the SAM, and CK response (more details are given below). The heterozygosis level of the F0S1–18 was not measured; however, F1S1–18 plants share at most two different alleles for each locus, since they are derived from a strict self-fecundation.

Studies of nucleotide polymorphism for genetic marker identification are in progress in our laboratories. Computational analyses of several KNOPE1 genes have not revealed, so far, any single nucleotide difference between ‘Chiripa’ and other cultivars (e.g. ‘Springcrest’), between the outdoor plants of the S1 and S7 lines, and within the sister plants of the F1S1–18 progeny. KNOPE1 was highly conserved at least in the transcribed and intron regions, and it was assumed that mechanisms of regulation would be conserved throughout the material used.

Plant material and sampling for KNOPE1 response to T. deformans infections
In the open field, 20 adult plants (S1 and S2 clones in plot 1) were treated yearly with two sprays of Bordeaux mixture (2%) and Thiram (0.4%) at leaf fall with a 2-week interval, followed by two sprays of Thiram (0.5%) in spring before bud swelling. Two rows of 10 adult clones (S1 and S2 clones in plot 2) were set at ~50 m from plot 1, and pesticide was only sprayed during the second of a 3-year interval (2001–2003). These plants exhibited curl disease symptoms typically from April to June, whereas the infection was rare on treated plants. In April–May, expanded leaves (mid-vein length ~8–10 cm) borne on distal positions of growing shoots were collected from healthy and infected trees. The infected samples exhibited red or green curly areas within or at the margins of leaves (see Fig. 5A), so that the pathogen damage was not yet extended. Northern blot, in situ localization, and immunolocalization experiments were performed on attacked and healthy leaves (n=3), which were sampled from three different S1 clones of the outdoor treated and untreated plots. Experiments were performed for 3 years (2001–2003).


Figure 5
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  		Fig. 5. The KNOPE1 pattern of expression. (A) Organ-specific expression by northern analysis. Organs examined are indicated above the lanes. Lamina 1 and 2 refer to expanded leaves bearing a major vein of ~8 cm and 12 cm, respectively. The KNOPE1 transcript size was ~ 1.6 kb as estimated by comparison with a co-migrating RNA marker. r-RNA, ethidium bromide-stained rRNA was photographed to check for equal loading. (B–D) In situ hybridization in vegetative meristems. The antisense probe 3 (Fig. 2A) was used and the purple stain indicates KNOPE1 mRNA. (B) Transverse and (C) longitudinal sections of the SAM at vegetative resumption. (D) Control reaction with a sense probe. ad, apical dome; dl, developing leaf; lp, leaf primordium CZ, PZ, RZ, central, peripheral, and rib zones; P0, site where the next leaf primordium will be formed; ps, procambial strands. Size bars: 35 µm in (B), (C), and (D).

 
Isolation and sequence analysis of cDNA and genomic clones
The cDNA was synthesized (see next paragraph) from herbaceous stem RNA of F1S1–18 plants and PCR-amplified by degenerate primers Kn5Fw [5'-GA(C/T)(C/G)A(A/G)TT(C/T)ATGGA(A/G)(A/G)C(A/G/C/T)TA-3'] and Kn8Bw [5'-AT(A/G)AACCA(A/G)TT(A/G)TT(A/T/G)AT(C/T)TG-3'], which were designed on DQEMEAY (KNOX2) and QINNWF (HD), respectively. A 482 bp fragment was cloned and sequenced, then the full-length KNOPE1 cDNA was rescued by 5'/3' RACE (rapid amplification of cDNA ends) technology (Invitrogen). Reverse 5' RACE primers for cDNA synthesis were: Kn4Bw (5'-GGAGGAGCATTATTGTTGCCAAGC-3') and Kn2Bw (5'-CTGCCTTGCCTCAAACTCC-3'), which yielded 916 bp and 734 bp fragments, respectively. 3' RACE primers were: cDNA synthesis oligo(dT) anchor and Kn7Fw (5'-CTCCACTTCGCATCTTCTCACCC-3'). PCR conditions were 500 ng of genomic DNA or 200 ng of cDNA, 1 mM of each primer, 0.5 mM dNTPs, 2.5 U of Taq DNA polymerase (TaqQUIA, Quiagen), 1/10 of 10x Taq buffer, 2.5 mM MgCl2, final volume 50 µl. Cycling conditions were an initial cycle at 95 °C for 5 min followed by 35 cycles at 95 °C for 40 s, 57 °C using cDNA or 62 °C using genomic DNA for 30–60 s, and 72 °C for 30–90 s, and a final extension at 72 °C for 5 min.

Introns were found by amplifying genomic DNA of F1S1–18 plant leaves with the following pairs of primers: Kn3Fw (5'-GCCAAGATCATCGCCCACCC-3')/Kn4Bw; Kn7Fw/Kn6Bw (5'-ATTCTCCTGCTCCTCTTCAG-3'); Kn11Fw (5'-AAGGTGGCATTGGCGGAGT-3')/Kn10Bw (5'-GCAGTTTGCTTTGTCTTTTGG-3'). The PCR product sequences were aligned to set intron size and locations. All PCR fragments were cloned into the pGEM-T easy vector system (Promega).

Yeast two-hybrid assay
KNOPE1 was amplified (cDNA fragment 226–1508) under the conditions reported above except using Taq Platinum Pfx (Invitrogen), and enzyme-adapted primers Kn1Fw (5'-GGGATCCGGATGGAAGAGTACAACC-3') and Kn10Bw (5'-GCTCGAGGCAGTTTGCTTTGTCTTTTGG-3'). The PCR product (50 ng) was ligated in BamHI–XhoI sites (underlined nucleotides) in the oriented direction into pENTR1A (Gateway system, Invitrogen), sequenced to check for gene integrity, and cloned into the TRP1-marked Gal4 activation domain vector (pDEST22, Invitrogen) through the LR reaction. Arabidopsis PNF (Atg27990), PNY (NM_120281), KNAT1/BP (NM_116884), STM (NM_104916 [GenBank] ), and BELL1 (NM_123506) were cloned into a LEU2-marked Gal4 DNA-binding domain construct (pDEST32) provided by Dr Enrico Magnani. Recombinant pDEST22 and pDEST32 were transferred into Saccharomyces cerevisiae (MaV103 and MaV203, respectively) by electroporation (1800 V for one pulse, EasyjectPrima-EQUIBIO) and then plated on selective medium. The recombinant colonies were used in mating-type procedures by plating on -LEU-TRP-deficient medium (YPAD). Plates were overlaid with chloroform, incubated for 5 min, and dried for 10 min under a hood, then X-Gal agarose (20 ml, 1% low-melting agarose, 0.1 M NaHPO4 buffer pH 7.0, 0.25 mg ml–1 X-Gal) was poured on the colonies and they were left to harden. Plates were incubated at 30 °C until a blue stain occurred. Inserts were also swapped from prey to bait vectors.

Genetic transformation of Arabidopsis
The 1–1508 bp KNOPE1 cDNA portion was amplified using adapted primers: Kn-5'UTRFw (5'-GTCTAGAGTTTGGCTTCACTTCCTCGC-3') and Kn-3'UTRBw (5'-GCTCGAGGCAGTTTGCTTTGTCTTTTGG-3'), and Taq Platinum Pfx (Invitrogen). PCR conditions were the same as above. The amplified fragment was digested with XbaI and XhoI, and cloned into the binary vector pBA002 (Kost et al., 1998) under the control of the 35S promoter of cauliflower mosaic virus (CaMV). The recombinant plasmid was sequence-checked and transferred to the Agrobacterium tumefaciens strain GV3101 (pMP90) by freeze–thaw (Holstein et al., 1978). The 35S:KNOPE1 was inserted in Arabidopsis Columbia by vacuum infiltration following Bechthold et al. (1998). Transformant lines were selected on medium (MS0 and Gambor vitamins) containing phosphinothricin, carbenicillin, and cefotaxime at 10, 500, and 90 ppm, respectively. The KNOPE1 transcript was detected by RT-PCR using Kn3Fw and Kn4Bw primers.

Alignments and phylogenetic analysis
The alignment of KNOPE1 with other KNOX proteins was performed by ClustalW (http://www.ebi.ac.uk.clustalw) and optimized by visual inspection (PILEUP program). Phylogenetic trees were constructed by MegaBlast2 (based on the minimum evolution criterion), using bootstrap values performed on 1000 replicates, and the 50% value was accepted as an indication of a well-supported branch. The class 1 KNOX accession numbers are: AtKNAT1, U14174; AtKNAT2, U14175; AtKNAT6L, AB072362; AtKNAT6s, AB072361; AtSTM, U32344; GmSBH1, P46608 [GenBank] ; HaKn1, AY096802; HaKn2, AY096803; HtKn1, CAD58394 [GenBank] ; InKN1, AB015999; InKn2, AB016000; InKN3, AB016002; LeT6, AF000141; LeTKn1, U32247; LetKN2, U76407; LeTKn3, U76408; LeTKn4, AF533597; MdKN1.1, Z71978; MdKN1.2, Z71979; MtKNOX1, AF308454 ; NtH1, AY169493; NtH9, AB025713; NtH15, BAA25546 [GenBank] ; NtH20, AB025714; NtH22, AB025715; NtKn1, AF544052; NtKn2, AF544053; PatARK1, AY755413; PbKn3, AAV49802 [GenBank] ; Pkn1, BAA31698 [GenBank] ; PsHOP1, AF063307; PsKn1, AAC32262 [GenBank] ; PsKN2, AF080105; PtdKn2, AY684937; PtdKn3, AY684938; and PtKN, AAV84585 [GenBank] . The class 2 KNOX proteins were used to create an outgroup; accession numbers: AtKNAT3, X92392; AtKNAT4, NP_196667 [GenBank] ; AtKNAT5, NP_194932 [GenBank] ; and AtKNAT7, AF308451.

Southern blot analysis
The technique was performed as previously described (Giannino et al., 2000). Filters were hybridized at 60 °C, washed twice (2x and 1x SSC/0.1% SDS) at 60 °C for 10 min, and exposed to Biomax films (Kodak) for 4–12 h at –80 °C. Probe 1 spanned the 780–1261 bp of KNOPE1 cDNA. Probe 2 spanned the 2796–3539 bp of KNOPE1 genomic sequence, in which HindIII cuts at nucleotide 2841.

Northern blot analyses
Total RNA was extracted according to Giannino et al. (2000) from 2-year-old F1S1–18 individuals (organ-specific expression) and S1 clones (response to T. deformans infection). RNA (10 µg per lane) was separated on a 1.2% (w/v) agarose–formaldehyde gel and transferred to a Hybond N+ membrane (Amersham Biosciences, UK). Hybridization was carried out overnight at 42 °C with Ultrahyb buffer (Ambion) containing formamide, followed by two washes in 2x and 1x SSC/0.1% (w/v) SDS at 60 °C for 10 min and one wash in 0.1x SSC/0.1% (w/v) SDS. Filters were exposed to Kodak BIOMAX films (Amersham Biosciences, UK) for at least 4 h at –80 °C. Probe 3 of KNOPE1 cDNA spanned the 1303–1681 bp stretch. The ISOPENTENYLTRANSFERASE 5 (IPT5) probe consisted of a 386 bp fragment, which was amplified by IPT5Fw (5'-GCGTGCCTGAAATGGATGAG-3') and IPT5Bw (5'-AGCGGTCACTGTCGGCATAG-3'), designed from the apple expressed sequence tag (EST) CN916352, and using leaflet cDNA. The single amplified product was cloned and sequenced, and the gene fragment was named IPT5 after Arabidopsis IPT5 beecause they shared 73% identity. The probes were radiolabelled with [32P]dCTP using the Ready Primer kit (Amersham Biosciences, UK).

RT-PCR analyses
RNA from 35S:KNOPE1 and wild-type Arabidopsis (Fig. 4A) was isolated by the TRIZOL reagent following the manufacturer's instructions (Invitrogen). For peach RNA, see the paragraph above. RNA was made DNA free (RQ1, Promega) and 3 µg was reverse transcribed (Superscript III, Invitrogen) at 55 °C into a single-stranded cDNA by oligo(dT)20. The KNOPE1 311 bp transcript was amplified by Kn3Fw/Kn4Bw primers; 26S rRNA by 26SFw (5'-AGCATTGCGATGGTCCCTGCGG-3')/26SBw (5'-GCCCCGTCGATTCAGCCAAACTCC-3'), and used to check for equal amounts of cDNA template. A mock PCR was performed using RNA samples to exclude DNA contamination. PCR trials were performed at distinct cycles to assess the variation of transcript abundance before signal saturation, at fixed primer pair and reaction parameters. Amplification products were sequenced to avoid artefacts. The PCR was conducted in a 50 µl total volume containing cDNA (3 µl); the chemical parameters are given above. Cycles were 35 for KNOPE1 and 20 for 26S rRNA; 15 µl was electrophoresed in a 1% agarose gel. Reverse-transcribed 26S rRNA appeared to be equal in all the tissues tested. In CK response assays (Fig. 7), the optical density (OD) of the signal bands was determined by the ID Image Analysis software (Kodak Digital Science), and the relative OD was graphed as histograms (Microsoft Excel) representing the ratio between the OD of KNOPE1 and 26S rRNA (checking for equal loading of RNA). RT-PCR experiments were carried out three times with independent RNA extracts. Standard errors were calculated and are indicated on the size bars. All data sets were subjected to Student's t-test where P <0.05 is regarded as significant.


Figure 4
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  		Fig. 4. Phenotypes of 35S:KNOPE1 Arabidopsis. (A) RT-PCR analysis. a and b, two distinct 35S:KNOPE1 T1 individuals; wt, wild type; actin 8 was used as a control for cDNA synthesis and loading. (B) Wild-type Columbia and (C) 35S:KNOPE1 Arabidopsis. (D) 35S:KNOPE1 leaf exhibiting lamina alteration and lobed margins and (E) leaflet-like outgrowths. (F) Wild-type and (G) 35S:KNOPE1 leaves devoid of chlorophyll highlight vascular modifications. Size bars: 0.4 cm in (B) and (C); 0.2 cm in (D); 0.2 mm in (E); 0.15 cm in (F) and (G).

 

Figure 7
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  		Fig. 7. Zeatin immunolocalization in leaves affected by curl disease. (A), (C), (E) Leaf type and position of sectioned areas (white box) for the zeatin immunolocalization experiments. Healthy (A) and infected (C, E) leaves. (B), (D), (F), (G), and (H) Zeatin (arrowed deep purple stain) in healthy (B) and affected leaves at S2 (D) and S3–S5 (F–H). (G) and (H) Magnifications of leaves at S3–S5 show zeatin accumulation in the epidermal and subepidermal (G) and mesophyll (H) host cells. uep, upper epidermis; pa, palisade layer; vsb, vascular bundle; sa, spongy aerenchima; se-h, subepidermal hyphae; sc-h, subcuticular hyphae; rc, reactive epidermal and subepidermal cells; mc, mesophyll cells. Size bars: 100 µm in (F); 75 µm in (B) and (D); 15 µm in (G) and (H).

 
Cytokinin response assay
Fully expanded leaves (mid-vein was ~8 cm) including the petioles were excised from the primary axis of 2-year-old F1S1–18 plants (June). They were immersed in sterile tubes filled with 50 ml of 0.1 µM NaOH (control) and 10 µM benzyl aminopurine (BAP)/0.1 µM NaOH. Samples were kept at 22 °C at a light intensity of 100 µmol m–2 s–1 PAR and removed from solutions after 0.5, 2, and 4 h, and frozen in liquid nitrogen. Non-immersed leaves, which were excised from the plant and rapidly frozen in liquid nitrogen, represented the controls at 0 h. RNA was isolated from a two leaf pool and the assay was repeated three times using leaves borne on three different plants.

In situ hybridization
Excised tissues were fixed, dehydrated, embedded in paraffin, cut into 8 µm sections, and hybridized (55 °C) to a digoxigenin-labelled antisense RNA probe as described by Cañas et al. (1994). The cDNA probe 3 was linearized by SpeI and NcoI. Antisense and sense probes were in vitro synthesized by T7 and SP6 polymerases, respectively (Giannino et al., 2000). In experiments performed on the SAM shoot, three apices were excised from 2-year-old F1S1–18 plants (n=3) at vegetative resumption (late February). In experiments of the KNOPE1 response during leaf curl disease, sections of attacked and healthy leaves (n=3) were sampled from three different S1 clones of the outdoor treated and untreated plots.

Cytological–histological analyses and zeatin immunocytolabelling
Both cytohistological procedures and zeatin immunolocalization were carried out using the same material as reported above. Concerning zeatin localization, the leaf portions were pre-embedded, cut with a vibratome (Leica VT1000E, Bensheim, Germany), and incubated with primary antibody against zeatin according to Dewitte et al. (1999). Colloidal gold- (<1 nm) labelled secondary antibodies (1:40, Aurion, Wageningen, The Netherlands) were used. The antibodies can recognize both the conjugated and free CK bases. However, the procedures of fixation (based on paraformaldehyde and glutaraldehyde mixtures) wash away the conjugated forms; hence, the antibodies specifically detected the free zeatin bases, which are biologically active (Dewitte et al., 1999).


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Supplementary data
 References
 
KNOPE1 is a class 1 KNOX-like gene
The full-length cDNA of KNOPE1 is 1681 bp (accession no. DQ358050; for details on cDNA features, see Supplementary Fig. S1 available at JXB online) with an open reading frame (ORF) of 1170 bp encoding a deduced polypeptide of 389 amino acids. KNOPE1 is a 44.41 kDa protein that contains the typical KNOX domains (Fig. 1A) and shares highest identity with Malus domestica KNAP1 (88%), followed by Populus spp. KN3 (73%), and A. thaliana KNAT1 (59%). The KNOPE1 HD was 100, 95.2, and 85.7% identical to those of apple KNAP1, poplar KN3, and maize KN1, respectively. The identity grade with the maize KN1 HD is a criterion to assign KNOX proteins to class 1 (Kerstetter et al., 1994). KNOPE1 fell into the monophyletic KNAT1/BP-like group, in a subclade of arboreal species (Fig. 1B); it was closest to apple KNAP1 (MdKN1). KNOPE1 and the other arboreal BP-like proteins shared highly conserved stretches at the N-terminus (e.g. RGNFLYAS, FHLQS, and VKTEA shaded in grey in Fig. 1A) not found in herbaceous KNOX proteins. Interestingly, a conserved VKT (position: 64–66) was maintained in six out of nine members of the BP-like group (LeTKN1, NtH20, PtdKN3, KNOPE1, MdKN1.1, and MdKN1.2 in Fig. 1B).


Figure 1
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  		Fig. 1. Features of KNOPE1 protein. (A) Alignment of the KNOPE1 deduced amino acid sequence (bold) with Populus tomentosa PtKN, Populus trichocarpaxP. deltoides PtdKN3, Malus domestica MdKN1.1 and MdKN1.2, and Arabidopsis thaliana KNAT1/BP. Gaps introduced for better alignment are shown by dashes; asterisks indicate strictly conserved residues. Grey shading encompasses amino acids conserved in tree species. The typical KNOX1, KNOX2, ELK domains, and the homeodomain (HD) are indicated. (B) KNOPE1 (bold) within the phylogenetic tree of class 1 KNOX dicot proteins. Class 2 KNOX proteins were used to create an outgroup. Bootstraps values (at the branching points) are given for major nodes and are based on 1000 replicates. Orthologous members of each subgroup are boxed and are named referring to the respective Arabidopsis KNOX.

 
To estimate the copy number of class 1 members in cultivar ‘Chiripa’, Southern blot analysis was initially performed with the cDNA probe 1 (Fig. 2A), which spanned the highly conserved KNOX2-HD stretch. The signal pattern (Fig. 2B, left panel) suggested the occurrence of a small gene family. Subsequently, probe 2 based on the KNOPE1 genomic sequence, which harboured a cleavage site for HindIII (Fig. 2A), was used. A single band was found (Fig. 2B, right panel) when genomic DNA was digested with EcoRI and EcoRV, whereas two signals were produced when using HindIII, and the ~1.8 kb band was consistent with the size predicted by restriction analysis (i.e. 1856 kb). This pattern strongly suggested that KNOPE1 was a single-copy gene.


Figure 2
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  		Fig. 2. KNOPE1 genome organization. (A) Scheme of KNOPE1, Zea mays (Zm) KN1, and Arabidopsis thaliana (At) KNAT1 genes. The black triangles and numbers indicate intron positions and sizes (in bp). Start and stop codons and the polyadenylation tail are indicated. The cDNA probe 1 spans the KNOX2-HD domains. The genomic probe 2 is KNOPE1 specific, including intron IV and the 3'-untranslated region. The genomic probe 3 proceeds downstream of the HD and was used for in situ experiments. Numbered and oriented arrowheads represent forward and reverse primers used to assemble the gene. H, HindIII site. (B) Southern blot analysis. Left panel, KNOX gene family: genomic DNA digested with ApaI, EcoRI, EcoRV, and XhoI, and hybridized with probe 1. Right panel: genomic probe 2 was used in combination with EcoRI, EcoRV, and HindIII; the latter cuts the probe once. The molecular weights of a co-migrating DNA marker are in kb.

 
KNOPE1 harboured four introns (Fig. 2A), which were flanked by the GT/AG editing motifs and rich in A/T bases. The first two introns lay in the KNOX1 and KNOX2 subdomains, maintaining the same positions as those of Arabidopsis KNAT1/BP and maize KN1 (Fig. 2A). The third intron was between KNOX2 and the HD, and the fourth fell within the HD, similar to the intron locations of the above-mentioned Arabidopsis and maize proteins. Finally, intron II of KNOPE1 (accession no. DQ388033) encompassed a stretch (position 907–917) which is conserved in introns II of monocot KNOX (Bauer et al., 2004).

KNOPE1 interacts with BELL1, PNF, and PNY, partners of Arabidopsis BP
To test the hypothesis that KNOPE1 and BP shared biochemical similarities, a yeast two-hybrid assay was carried out (Fig. 3). The interactions were tested using S. cerevisiae harbouring the Arabidopsis genes PNF, PNY, STM, BELL1, and BP as prey for the KNOPE1 bait. Blue signals indicated the binding of KNOPE1 to BELL1, PNF, and PNY, whereas null staining indicated a lack of interaction of KNOPE1 with KNAT1, STM, KNOPE1 itself, and empty vectors (data not shown). Notably, the KNOPE1 interaction pattern was essentially the same as that of BP. Experiments with swapped pray and bait were performed and the pattern described above did not vary (data not shown).


Figure 3
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  		Fig. 3. Yeast two-hybrid assay. Prey proteins KNOPE1 (left column) and BREVIPEDICELLUS (BP, right column), and Arabidopsis baits POUNDFOOLISH (PNF), PENNYWISE (PNY), BELL1, SHOOT MERISTEMLESS (STM), BP, and KNOPE1 itself were expressed in S. cerevisiae. The blue staining (here rendered in dark grey) indicates the occurrence of a prey–bait interaction.

 
35S:KNOPE1 and 35S:BP Arabidopsis transgenics share common altered traits
The A. thaliana ecotype Columbia was transformed with the full-length KNOPE1 driven by the CaMV 35S promoter. RT-PCR analyses (Fig. 4A) confirmed KNOPE1 expression in the transgenics. Several independent T1 lines (an example is shown in Fig. 4C) exhibited alterations in leaf morphology compared with controls (Fig. 4B). The shape of the first two leaves of 35S:KNOPE1 plants was unaltered, whereas the rosette leaves showed lamina alteration and lobed margins (Fig. 4D) accompanied by sporadic leaflet-like outgrowths (Fig. 4E). There was also pronounced thickening of the central rib and a reduction or loss of dominance of the central vein with secondary ones (Fig. 4F, G). These traits were similar, if not identical, to those observed in the 35S:BP Arabidopsis (Lincoln et al., 1994).

In peach, KNOPE1 is down-regulated in leaf primordia flanking the vegetative meristems
Northern analyses and in situ hybridization were used to study the strength of spatial expression patterns of KNOPE1 (Fig. 5). In aerial vegetative organs (Fig. 5A, left panel, lanes 2–4), the message was most abundant in herbaceous stems of young shoots (May), followed by apical tips (apices and leaflets), and pre-shooting vegetative buds. In leaves (Fig. 5A, central panel), the message occurred in petioles, but was undetected in the laminas. In reproductive organs (Fig. 5A, right panel), the transcript signal was intense in swollen floral buds inclusive of pedicels (February), faint in pistil/ovaries and sepals, and absent in stamens and petals of open flowers (March). Finally, KNOPE1 expression was also observed in seedling roots (Fig. 5A, left panel, lane 1).

In transverse sections of vegetative SAMs (Fig. 5B), the KNOPE1 message (purple stain) marked the apical dome (ad), but was absent in developing leaflets (dl). In longitudinal sections (Fig. 5C), it was absent in the tunica, but abundant in the corpus and in the boundaries at the base of leaf primordia. More precisely, the transcript featured in the PZ, but not in the site where the next leaf would form (P0), in the RZ, and procambial strands (ps). In controls, a sense probe failed to produce a signal (Fig. 5D).

KNOPE1 is triggered and its mRNA differentially localized at distinct stages of curl disease
Northern analyses indicated that KNOPE1 and IPT5 were up-regulated in leaves naturally infected by the fungus and exhibiting distortions (Fig. 6A). To monitor KNOPE1 mRNA localization during the disease, reference was made to (i) the model of Syrop (1975a, b), who defined the T. deformans developmental stages (S1–S5) associated with cytohistological modifications of almond leaf, and (ii) histological data from peach leaf curl disease (Bassi et al., 1984). Table 1 shows a synopsis of T. deformans developmental stages, and symptoms and histological features of infected leaves, and correlates them to the respective panels in Fig. 6. Tissues used for in situ hybridizations were: sectors of healthy leaves (Fig. 6B) at S1 (Fig. 6C), healthy sectors without distortions of infected leaves (Fig. 6E) sited at the border of curly areas, at S2 (Fig. 6F–H), and curly sectors of infected leaves (Fig. 6J) at S3–S5 (Fig. 6K). In the lamina of healthy expanded leaves, the KNOPE1 transcript was undetected in all cell types (Fig. 6D). At S2, the KNOPE1 message (Fig. 6G–I) localized uniquely to the cells of the palisade layer (pa), whereas it spread in a scattered manner (Fig. 6L) at S3–S5, including the vascular bundles (vsb) and the spongy parenchyma (sa).


Figure 6
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  		Fig. 6. KNOPE1 response in leaves affected by curl disease. (A) Healthy (left) and infected (right) leaves are photographed (major vein of ~8 cm). In northern blots, the RNA was derived from a three-leaf pool, and the pictured results are typical of three distinct experiments. The KNOPE1 and IPT5 transcript sizes are indicated in kb. r-RNA, ethidium-bromide-stained rRNA. (B), (E), (J) Leaf type and position of sectioned areas (white box) for the in situ experiments. Healthy (B) and infected (E, J) leaves. (C), (F), (H), (K) Toluidine blue-stained sections of healthy (C) and infected leaves showing fungal stages S2 (F and the magnification H) and S3–S5 (K). (D), (G), (I), (L) KNOPE1mRNA (visible as purple stain) in healthy (D) and infected leaves at S2 (G and the magnification I), and at S3–S5 (L). uep, upper epidermis; pa, palisade layer; vsb, vascular bundle; sa, spongy aerenchyma; cu, cuticle; alter. and org. pa, altered palisade with isodiametric cells and organised palisade with elongated cells, respectively; se-h, subepidermal hyphae; sc-h, subcuticular hyphae; ah, ascogenous hyphae; as, ascospores. Size bars: 15 µm in (H); 20 µm in (F) and (K); 25 µm in (I); 50 µm in (C), (D), (G), and (L).

 

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  		Table 1. Synopsis of Taphrina deformans developmental stages, and symptoms and histological features of infected leaves

 
Zeatin accumulation is a host response associated with histological disorders of leaf curl disease
The triggering of IPT5 (Fig. 6A) and the histological alteration of curly areas suggested the occurrence of changes in CK homeostasis. Hence, zeatin immunolocalization was performed on tissue sectors (Fig. 7A, C, E) consistent with those where KNOPE1 in situ hybridization was carried out (Fig. 6D, G, L). Zeatin mainly localized in the vascular bundles (vsb) and in sporadic mesophyll cells of both uninfected leaves (Fig. 7B) and green sectors of affected leaves (Fig. 7D). In contrast, an intense signal spread diffusely in curly hyperplastic sectors (Fig. 7F) and, at high magnifications, the zeatin signal was observed inside the host cells (rc) interacting with subepidermal mycelium (Fig. 7G, H). Notably, the anti-zeatin antibodies cross-hybridized with the subcuticular hyphae (sc-h) and ascogenous cells (not shown), but not with subepidermal hyphae (se-h), suggesting that T. deformans was able to produce CK-like compounds at late developmental stages (Johnston and Trione, 1974). As for controls, tissues were hybridized with a sense probe in the in situ experiments, whereas immunoreaction was performed without primary anti-zeatin antibody in immunolocalization experiments. In both cases, signal above background was not detected (see Supplementary Fig. S2 at JXB online).

Leaf KNOPE1 transcription is prompted by cytokinin treatment.
To ascertain KNOPE1 responsiveness to CK, expanded leaves were immersed in 10 µM BAP and transcription was monitored in a time lapse of 0.5, 2, and 4 h (Fig. 8A). A mock PCR and water control reactions were performed and no signal occurred (not shown). A very faint signal of KNOPE1 mRNA was observed in non-immersed control leaves (Fig. 8A, lane 0 h), very probably due to gene activity in petioles (see Fig. 5A), which were included in the sampling of the experiments. In leaves soaked in BAP-free solution, KNOPE1 expression increased after 0.5 h, but fell below the level of non-immersed leaves within 4 h (Fig. 8B, white histograms), suggesting that a response to stress effects (e.g. anoxia) may have occurred. The message abundance of BAP-treated leaves was higher than that of non-immersed leaves, and the transcriptional increment was maintained from 0.5 h onwards (Fig. 8B, grey histograms). Consequently, KNOPE1 mRNA was significantly more abundant in leaves treated with BAP than in those soaked in BAP-free solution during the whole time lapse, suggesting a specific response to the CK treatment.


Figure 8
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  		Fig. 8. KNOPE1 response in cytokinin-treated leaves. (A) RT-PCR for KNOPE1 in leaves immersed in a 6-benzyl aminopurine (6-BAP) solution and sampled after 0.5, 2, and 4 h. Non-immersed leaves (0 h) and leaves soaked in 6-BAP free solution were used as controls. The 26S was used as control for the amplification. (B) Relative optical density to estimate KNOPE1 message abundance with respect to 26S. Analyses were performed three times with independent RNA extracts (each deriving from two leaf pools). Bars indicate standard errors and the gel bands represent one of the experiments.

 

   Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Supplementary data
 References
 
The peach KNOPE1 full-length cDNA was isolated, and its deduced protein belonged to class 1 KNOX, falling in a clade of the BP-like group. The gene is likely to be a single copy within a family of four or five class 1 members in the ‘Chiripa’ genome. Just like the maize and Arabidopsis orthologues, it is organized into five exons and four introns, and did not harbour any intron inside the ELK, another typical feature of class 1 KNOX (Reiser et al., 2000). Intron II included a stretch found in introns II of maize and rice class 1 genes (Bauer et al., 2004). This motif might have been selected for a common function throughout monocot and dicot KNOX.

Protein–protein interaction assays indicated that KNOPE1 bound to a set of Arabidopsis BELL1-like proteins, which also heterodimerized with BP (Smith et al., 2003). KNOPE1 overexpression in Arabidopsis caused mutant phenotypes, which were highly similar to 35S:BP Arabidopsis lines. KNOPE1 transcript localization in peach SAM strongly resembled that of BP in Arabidopsis (Lincoln et al., 1994). KNOPE1 mRNA was undetected in SAM P0 cells, and leaf primordia (Fig. 5C) and lamina of expanded leaves (Figs 5A, 6D), consistent with the idea that class 1 KNOX down-regulation is a key step in leaf initiation and organogenesis and that the exclusion of KNOX proteins from leaves results in a simple leaf shape (Hay and Tsiantis, 2006).

Organs containing tissues with meristematic activity show KNOPE1 expression (Fig. 5A) and most of them (including roots, see Truernit et al., 2006) coincide with those of Arabidopsis in which BP is active (Lincoln et al., 1994; Venglat et al., 2002). Intriguingly, KNOPE1 was detected in pedicels of expanded leaves (Fig. 5A). BP signal is absent in the petioles of Arabidopsis leaves (Hay and Tsiantis, 2006), though found at the petiole base in other works (Ha et al., 2003), and constantly present as a ring encircling the stem leaf traces (Douglas and Riggs, 2005). Petioles of peach leaves harbour a wide vascular bundle, characterized by an active cambium, and undergo lignification. In our laboratories, KNOPE1 transcript was detected in both the intrafascicular cambium and phloem-associated cells of growing petioles (unpublished results), suggesting that KNOPE1 may participate in maintaining the meristem identity of cambium cells.

Interestingly, KNOPE1 expression was triggered in expanded leaves attacked by T. deformans, and the message occurred specifically in the palisade cells invaded by the fungus at early stages of development (S2; Syrop, 1975a). At S2, host cell de-differentiation and division are not observed, though several modifications rapidly occur at the subcellular level (Syrop, 1975b). The KNOPE1 message localization to the palisade—but not to the epidermal cells—suggests that a programme of indeterminate cell fate is established in specific cell types. At later fungal stages (S3–S5), numerous dividing isodiametric cells (hypertrophy) replace the leaf palisade layer, and few other cells undergo hyperplasia, both leading to tissue distortions (Bassi et al., 1984). The chaotic misexpression of KNOPE1 may be responsible for the maintenance of cell indefinite identity, thus preventing further differentiation. In this context, KNOPE1 behaviour is consistent with that of class 1 KNOX when these are overexpressed in leaves (Scofield and Murray, 2006).

KNOX up-regulation can induce CK synthesis de novo by triggering plant IPT genes (Jasinski et al., 2005; Sakamoto et al., 2006), which act to increase zeatin production (Sakakibara, 2006). Hence, it is salient that the peach IPT5 was up-regulated in infected leaves. The present experiments showed a higher number of zeatin-immunoreactive mesophyll cells in curly sectors than those from both uncurled areas of attacked leaves and healthy leaves. Interestingly, zeatin was localized inside the host cells. Such an event, together with the IPT triggering, indicated that zeatin accumulation was a plant response. The role of CKs in comparable plant–biotroph interactions is complex. A host CK increase may promote (Walters and Mc Roberts, 2006) nutrient sink and senescence delay, but favour suppression of rapid host cell death (hypersensitive response). However, a high CK concentration is also reported to induce cell necrosis (Carimi et al., 2003). A mutual interaction underlies class 1 KNOX genes and CK (Rupp et al., 1999; Sakamoto et al., 2006), and KNOPE1 was triggered in leaves treated with BAP, probably due to a specific response from petiole cambium cells. Consequently, at later stages of fungus development, KNOPE1 and zeatin may share reciprocal (up) regulations to induce and maintain meristematic identity, thus preventing cell differentiation.

Taphrina deformans is an obligate biotrophic fungus, and in vitro-based infections have been unsuccessful (see Supplementary Fig. S3 at JXB online). Although the occurrence of other pathogens (bacteria, viruses, and mycoplasms) could not be absolutely excluded, histological analyses allowed monitoring and the fungus movement/stages could be strictly related to the host responses. On this basis, it is proposed that KNOPE1 expression in the palisade may belong to an early response process occurring when leaf cells interact with subepidermal hyphae. KNOPE1 activation may mark the start of indefinite cell fate programmes and cause the IPT up-regulation that leads to zeatin overproduction (Jasinski et al., 2005). After the onset of hyperplasia, KNOPE1 and zeatin may establish a mutual interaction that maintains the abnormal leaf development. This model requires further experiments, dissecting the overlapping pathways in which KNOPE1 is involved during the disease course.


   Supplementary data
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary data
References
 
Figure S1. Features of KNOPE1 cDNA.

Figure S2. Control experiments of Fig. 6.

Figure S3. Information on Taphrina deformans infection.


   Acknowledgements
 
We are grateful to Mr Luigi Santini for technical support, and Dr Enrico Magnani (Department of Plant and Microbial Biology, Berkeley, CA, USA) for providing two-hybrid assay constructs. This work was supported by the EU project FAIR CT 96-1445 and CNR.


   Footnotes
 
* These authors contributed equally to the work. Back


   References
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 Abstract
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 Materials and methods
 Results
 Discussion
 Supplementary data
 References
 
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