The ‘Candidatus Phytoplasma mali’ effector protein SAP11CaPm interacts with MdTCP16, a class II CYC/TB1 transcription factor that is highly expressed during phytoplasma infection

Cecilia Mittelberger; Bettina Hause; Katrin Janik

doi:10.1371/journal.pone.0272467

. 2022 Dec 15;17(12):e0272467. doi: 10.1371/journal.pone.0272467

The ‘Candidatus Phytoplasma mali’ effector protein SAP11_CaPm interacts with MdTCP16, a class II CYC/TB1 transcription factor that is highly expressed during phytoplasma infection

Cecilia Mittelberger ¹, Bettina Hause ², Katrin Janik ^1,^*

Editor: Keith R Davis³

PMCID: PMC9754288 PMID: 36520844

Abstract

’Candidatus Phytoplasma mali’, is a bacterial pathogen associated with the so-called apple proliferation disease in Malus × domestica. The pathogen manipulates its host with a set of effector proteins, among them SAP11_CaPm, which shares similarity to SAP11_AYWB from ’Candidatus Phytoplasma asteris’. SAP11_AYWB interacts and destabilizes the class II CIN transcription factors of Arabidopsis thaliana, namely AtTCP4 and AtTCP13 as well as the class II CYC/TB1 transcription factor AtTCP18, also known as BRANCHED1 being an important factor for shoot branching. It has been shown that SAP11_CaPm interacts with the Malus × domestica orthologues of AtTCP4 (MdTCP25) and AtTCP13 (MdTCP24), but an interaction with MdTCP16, the orthologue of AtTCP18, has never been proven. The aim of this study was to investigate this potential interaction and close a knowledge gap regarding the function of SAP11_CaPm. A Yeast two-hybrid test and Bimolecular Fluorescence Complementation in planta revealed that SAP11_CaPm interacts with MdTCP16. MdTCP16 is known to play a role in the control of the seasonal growth of perennial plants and an increase of MdTCP16 gene expression has been detected in apple leaves in autumn. In addition to this, MdTCP16 is highly expressed during phytoplasma infection. Binding of MdTCP16 by SAP11_CaPm might lead to the induction of shoot proliferation and early bud break, both of which are characteristic symptoms of apple proliferation disease.

Introduction

’Candidatus Phytoplasma mali’ (’Ca. P. mali’) is the bacterial pathogen associated with the so-called apple proliferation disease [1]. Apple trees affected by this disease show different growth aberrations like uncontrolled shoot proliferation or enlarged stipules, early bud break and flowering and so-called witches’ brooms. Symptomatic trees produce small, tasteless, and unmarketable fruits, which causes economic losses and threatens apple production [2]. Phytoplasmas are wall-less, phloem-limited bacteria, that need insect vectors for their transmission and use effector proteins to manipulate their host plants [3–6]. ’Ca. P. mali’ is transmitted by certain psyllids [7] and encodes several potential effector proteins that appear to be released into Malus plants by a Sec-dependent secretion system [8,9]. So far, the best characterized effector protein is SAP11_CaPm, which shows similarity to SAP11_AYWB from ’Candidatus Phytoplasma asteris’ that is associated with Aster Yellow Witches’ Broom disease in aster [10]. Despite of being the best described effector, SAP11_CaPm’s function is not fully unraveled yet, and there are still knowledge gaps regarding its interaction partners in Malus × domestica, the natural host of ’Ca. P. mali’.

The development of witches’ brooms, i.e., uncontrolled branching, is a common symptom not only for apple proliferation, but also for several other phytoplasma diseases. Overexpression of SAP11_AYWB and SAP11-like proteins from other phytoplasma species resulted in an increased formation of lateral shoots [11–15]. Interestingly, SAP11_AYWB, SAP11_CaPm and other SAP11-like proteins bind and destabilize different TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR 1 and 2 (TCP) transcription factors [12,14,16].

TCP transcription factors are highly conserved in all land plant lineages and contain a basic helix-loop-helix (bHLH) domain, responsible for DNA binding [17]. TCPs are classified according to sequence differences in the bHLH-domain in class I and class II subfamilies [18]. The class II TCPs are further subdivided in CINCINNATA (CIN)-like TCPs and in CYCLOIDEA/TEOSINTE BRANCHED1 (CYC/TB1)-like TCPs [19]. TCPs of both classes play a key role in morphological development of plants, stress adaptions and plant immunity [20] and are thus interesting targets of diverse pathogen effector proteins [21,22].

A total of 52 TCP-domain containing genes were identified in apple [23]. For CIN-like class II TCPs, MdTCP25 (orthologue to AtTCP4) and MdTCP24 (orthologue to AtTCP13), an interaction with SAP11_CaPm has been shown [8]. Additionally, a yeast-two-hybrid (Y2H) screen using SAP11_CaPm revealed cDNA fragments of MdTCP16, a AtTCP18-like CYC/TB1 class II TCP and of the putative chlorophyllide b reductase NYC1 as putative interaction partners [8]. An interaction between SAP11_CaPm and the corresponding full-length gene products of the MdTCP16- and MdNYC1-fragment could not be confirmed at that time. Shortly after, the genome of Malus × domestica was de novo assembled and updated [24]. This sheds new light on the full-length ORFs including MdTCP encoding genes and led to some new sequence and reading-frame information regarding the assigned full-length genes of MdTCP16 and MdNYC1.

AtTCP18 is a key regulator of shoot branching [25–27] and is also known as BRANCHED1 (BRC1). BRC1 is used as a common term for all AtTCP18 orthologues in other plant species. Its expression is repressed by cytokinin, gibberellin, phytochrome B and sugar, and is promoted by auxin, an important regulator of apical dominance, strigolactones and a low red to far-red light ratio [28]. BRC1 influences not only the plant architecture, but also the seasonal growth of perennial plants, such as temperate fruit trees by responding to photoperiodic changes [29]. In addition, BRC1 interacts with the FLOWERING LOCUS T protein in Arabidopsis and represses floral transition in axillary meristems [30].

Since several other SAP11-like proteins interact with AtTCP18-like TCPs [11,13,15,31,32] and SAP11_CaPm from ’Ca. P. mali’ strain PM19 showed an interaction with AtTCP18 in an Y2H screen [16], the aim of this study was a detailed analysis of the two potential interactions between SAP11_CaPm with either MdTCP16 (i.e. the orthologue of AtTCP18) or MdNYC1. Moreover, to gain a better understanding of the role of MdTCP16, MdTCP24 and MdTCP25 in the host plant during phytoplasma-infection, the expression of these TCPs was determined by qPCR analysis of non-infected and infected Malus × domestica leaf samples in spring and autumn.

Materials and methods

Yeast-two-hybrid screen

In a previous study cDNA sequences that were partially similar to genes encoding MdTCP16 and MdNYC1 were identified as interactors of SAP11_CaPm from ’Ca. P. mali’ strain STAA (Accession: KM501063) by a Y2H screen [8]. These cDNA nucleotide sequences were obtained by sequencing the prey vectors in the identified yeast colonies and blasting against the NCBI nt-database [33,34]. The sequence, identified as MdTCP16, shared 100% sequence identity with the reference sequence XM_008376500.2 and covers 69.5% of the coding sequence for protein XP_008374722.1 (S1 Fig). Primers were designed to amplify the coding sequence of XP_008374722.1 (S1 Table). These primers contained overhangs that attached SfiI-restriction sites to the amplicon for subsequent cloning into the pGAD-HA Y2H prey vector.

The sequence identified as MdNYC1 shared 100% sequence identity to the reference sequence XM_029109831.1 and covers 13.7% of the C-terminal end of XP_028965664.1. Primers were designed to amplify the coding sequence of XP_028965664.1 and of the C-terminal part identified as an interactor of SAP11_CaPm in the Y2H screen. Both amplicons were subcloned into pGAD-HA prey vector via their primer-attached SfiI-overhangs.

The Y2H prey vector was co-transformed with pLexA-N-SAP11_CaPm bait vector into the Saccharomyces cerevisiae strain NMY51 cells [8,35]. Growth was monitored four days after transformation on selective SD plates lacking the amino acids adenine, leucine, histidine and tryptophane.

Bimolecular fluorescence complementation analysis

Primers, specific for MdTCP16 and SAP11_CaPm from ’Ca. P. mali’ strain STAA and with attB-site overhangs were designed (S1 Table) and fragments were amplified in a total of 50 μL reaction volume using 10 ng template and a final concentration of 1 x iProof HF Buffer, 200 μM dNTPs (50 μM each nucleotide), 0.5 μM of each primer and 0.02 U/μl iProof DNA Polymerase (Bio-Rad, Hercules, CA). The cycling conditions were 30 sec of initial denaturation at 98°C followed by 35 cycles of 98°C for 10 sec, 60°C for 30 sec and 72°C for 90 sec. The final extension was carried out at 72°C for 5 min. The amplified fragments were analyzed on a 1.2% agarose-gel and extracted with Kit Montage Gel Extraction columns (Millipore, Bedford, MA). The purified amplicons were used in a BP-reaction for the creation of Gateway-Entry vectors using 1 μL purified amplicon, 100 ng of donor vector (pDONR221-P1P4 or pDONR221-P3P2), 1 μL BP Clonase™ II enzyme (Invitrogen, Carlsbad, CA) following the manufacturer’s protocol. Depending on the N- or C-terminal location of the split-YFP in pBiFC vectors [36], MdTCP16 and SAP11_CaPm entry-vectors with or without stop-codon were combined in a LR-reaction using Gateway™ using LR Clonase™ II enzyme mix (Invitrogen) and following the manufacturer’s instructions.

Nicotiana benthamiana leaf mesophyll protoplasts were isolated for bimolecular fluorescence complementation analysis (BiFC) from leaves of four-week-old plants and protoplasts were transformed with the pBiFC vectors as described in Janik et al. (2017) [37]. In detail, leaf pieces of two N. benthamiana leaves were vacuum infiltrated for 30 min with 10 mL of enzyme solution containing 0.4 M mannitol, 20 mM KCl, 20 mM 4-morpholineethanesulfonic acid (MES), 1.5% (w/v) cellulase R-10, 0.4% (w/v) macerozyme R-10, 10 mM CaCl₂, and 1 mg/mL bovine serum albumin. The leaf pieces were then incubated in the dark at room temperature for 4 h and afterwards gently shaken for 30 min in the dark to release the protoplasts. The protoplast-leaf pieces solution was filtrated through a cell strainer into two 15 mL round bottom centrifuge tubes and centrifuged at 200 x g and 4°C for 1 min. The supernatant was carefully removed, and the first pellet was resuspended in 3 mL buffer W5 (154 mM NaCl, 125 mM CaCl₂, 5 mM KCl, 2 mM MES at pH 5.7). The second pellet was carefully resuspended with the first suspension. The protoplast suspension was incubated in the dark on ice for 40 min for sedimentation of the protoplasts. The protoplast pellet was again carefully resuspended in 3 mL buffer W5 and incubated for another 40 min in the dark on ice. The protoplast pellet was resuspended in 2 mL buffer MMG (0.4 M mannitol, 15 mM MgCl₂, 4 mM MES at pH 5.7) and protoplast density was counted in a Neubauer improved cell counting chamber. The protoplast solution was diluted with buffer MMG to a final concentration of 100,000 protoplasts per mL. 10 μg of pBiFC vector plasmid DNA, obtained by plasmid preparation with NucleoSnap Plasmid Midi kit (Macherey-Nagel, Düren, Germany) followed by a PEG precipitation, were mixed with 200 μL of protoplast solution. 220 μL of PEG-transformation-solution (0.2 M mannitol, 0.1 M CaCl₂, 40% (w/v) PEG 4000) were added to the protoplast-DNA mix and mixed by carefully shaking the tube. After 5 min of incubation at room temperature, 880 μL of buffer W5 were carefully added to the suspension. The protoplast transformation solution was centrifuged for 1 min at 4°C and the pellet was resuspended in 200 μL buffer WI (0.5M mannitol, 20mM KCl, 4mM MES). The tubes were then placed horizontally in the dark for incubation at room temperature overnight. Transformed protoplasts were analyzed 16h after transformation using a confocal laser scanning microscope (Zeiss LSM800, Carl Zeiss Microscopy, Oberkochen, Germany). Transformation rate and BiFC rate were determined as a mean value of three independent repetitions of protoplast transformation.

Plant material

Trees in the greenhouse: Apple rootstocks M9 were graft inoculated with ’Ca. P. mali’ strain PM6 (AT2-subtype) [38] infected or non-infected control scions of Malus x domestica cultivar (cv.) Golden Delicious and kept in greenhouse without temperature or light control. These small, one-year-old plants had a maximum height of about 40 cm.

Trees in the foil tunnel: Fully grown, ca. 2 m high, naturally infected Malus x domestica cv. Golden Delicious trees were grown and kept in an insect safe foil tunnel. Trees were infected with the locally predominant AT2 strain as determined by Sanger sequencing. Since it was a natural infection it cannot be ruled out, however, that a mixed infection with other strains was present in these trees. Nevertheless, this situation resembles the one in the field. Leaf samples for the analyses were taken in May and October 2011 as described in Janik et al. 2017 [8]: For each time-point pools of leaves from non-infected and infected trees (eleven trees/pool in May and six trees/pool in October), respectively, were assembled. Each of the pools of infected trees comprised material from trees representing the same symptom intensities. Equal amounts of material from each tree were pooled.

The phytoplasma levels from pooled leaf and root samples are reported in S2 Fig.

RNA extraction and cDNA synthesis

Greenhouse samples were collected in May and October 2021 from single apple trees. Leaf discs from five non-infected and five infected, one year old apple trees, grown in greenhouse were excised, and immediately frozen in liquid nitrogen. Leaf discs were grinded with mortar and pestle under liquid nitrogen. 100 mg of leaf powder was used for RNA extraction following protocol A of the Spectrum™ Plant Total RNA Kit (Sigma-Aldrich, St. Louis, MO). RNA was eluted in 50 μL elution buffer. RNA concentration was measured with a Spectrophotometer (NanoPhotometer® N60, Implen, München, Germany). 1 μg or 2 μg of RNA were reverse transcribed into cDNA by using SuperScript™ IV VILO™ Master Mix with ezDNase enzyme (Invitrogen), following the protocol that includes gDNA digestion with DNase enzyme. Samples that contained all reagents and RNA except the reverse transcriptase (“No-RT controls”) were performed in parallel. Synthesized cDNA was stored at -80°C.

Sampling of naturally infected trees of Malus × domestica cv. Golden Delicious was done in May and October 2011. Three pools of leaves from control or ’Ca. P. mali’-infected trees (6–11 trees/pool) were tested. RNA extraction and cDNA preparation is described in Supplementary Material and Methods of Janik et al. (2017) [8]. Synthesized cDNA was stored at -80°C.

DNA extraction

DNA of Malus × domestica leaf samples was extracted from approx. 50 mg grinded leaf tissue, using the DNeasy Plant Mini Kit (Qiagen, Venlo, The Netherlands), following the manufacturer’s instruction. DNA was eluted in 50 μL elution buffer.

qPCR analysis

The transcription factors MdTCP16, MdTCP25, MdTCP24 and the effector protein SAP11_CaPm were amplified with specific qPCR primer pairs (S1 Table) using a SYBR-Green qPCR assay. Reactions were run in a total of 10 μl, using 2 x Universal KAPA SYBR^® FAST master mix (KAPABIOSYSTEMS, Wilmington, MA), 20 pmol of forward and reverse primer and 2 μl of 1:50 diluted cDNA samples. Additionally, qPCR master mixes for the reference genes GAPDH, tip41 and EF1α were prepared for all samples, using the same reagent and template concentrations, and cycling conditions as mentioned above. All targets were amplified on the same 384well plate in one qPCR run on a CFX384 Touch Real-Time PCR Detection System (Bio-Rad), using three technical replicates per sample. Cycling conditions were as follows: 95°C for 20 sec, followed by 35 cycles with 95°C for 3 sec and 60°C for 30 sec, melt curve from 65°C to 95°C with an increment of 0.5°C/5 sec.

For determining the qPCR efficiency of every primer-combination, a four-point dilution series (1:10, 1:50, 1:100, 1:200) of a cDNA sample mixture was analyzed in each qPCR run.

Phytoplasma quantity was determined by the detection of the phytoplasma specific 16S gene together with the Malus specific single copy gene ACO as described by Baric et al. (2011) [39]. In brief, 2 μl of template DNA were analyzed in a total multiplex reaction volume of 20 μl, using 2x iQ^TM supermix master mix (Bio-Rad), 18 pmol of each qAP-16S forward and reverse primer, 4 pmol qAP-16S probe, 4 pmol of each qMD-ACO forward and reverse primer and 4 pmol of qMD-ACO probe. The ’Ca. P. mali’ specific qAP-16S probe was 5’-labeled with the reporter dye FAM, while the Malus specific qMD-ACO probe was 5’-labeled with VIC. Cycling conditions were as follows 95°C for 3 min followed by 35 cycles of 95°C for 15 sec and 60°C for 60 sec. Reactions were run in triplicates on a CFX96 Touch Real-Time PCR Detection System (Bio-Rad). For determining the qPCR efficiency, a five-point dilution series (undiluted, 1:10, 1:50, 1:100, 1:200) of a sample mixture of DNA from infected Malus × domestica roots was analyzed in each qPCR run.

Data analysis

Normalized expression was calculated according to Taylor et al. (2019) [40] considering qPCR efficiency (E) of each run, following the formula 1+E^ΔCq for relative quantity. Statistical analysis was performed with GraphPad Prism 7.05 (GraphPad Software Inc., La Jolla, CA).

Results

Yeast-two-hybrid and bimolecular fluorescence complementation analyses

The Y2H test using SAP11_CaPm from ’Ca. P. mali’ strain STAA fused to the GAL4-binding domain as bait revealed an interaction between MdTCP16 and SAP11_CaPm. An interaction between SAP11_CaPm and MdNYC1 (neither the fragment nor the full-length protein) was not detectable (Fig 1).

Fig 1 — Interaction between the bait SAP11_CaPm fused to a DNA binding domain (BD) and the prey protein from *Malus* × *domestica* fused to an activation domain (AD) is indicated by growth of the *Saccharomyces cerevisiae* reporter strain NMY51 on SD minimal medium lacking the amino acids histidine (his) and adenine (ade). The white color of BD-SAP11_CaPm + AD-MdTCP16 grown on full medium is an additional indication for a strong interaction, since the ADE2 reporter gene is activated upon interaction, while in absence of a protein-protein interaction and thus no ADE2 activation, a red colored intermediate accumulates in the adenine metabolic pathway.

For further confirmation of the interaction between SAP11_CaPm and MdTCP16, the sequences of SAP11_CaPm and MdTCP16 were subcloned into different pBiFC-2in1 vectors [36] by Gateway-cloning [41]. The pBiFC-2in1 vectors were transformed into N. benthamiana mesophyll protoplasts [37] and interaction was analyzed by confocal microscopy 16 h after transformation. Up to 92.5% of the transformed protoplasts showed a YFP signal, resulting from the interaction between SAP11_CaPm and MdTCP16 (Fig 2). The YFP-signal was localized to the cell nucleus and occurred additionally in the cytoplasm. The co-expression of SAP11_CaPm and MdNYC1 fused to both YFP-halves did not reveal YFP fluorescence after transformation of protoplasts, confirming the Y2H results (Fig 2).

Fig 2 — *Nicotiana benthamiana* mesophyll protoplasts were co-transformed with a BiFC expression vector encoding SAP11_CaPm and MdTCP16. SAP11_CaPm and MdTCP16 interact in the nucleus and in the cytoplasm as indicated by the occurrence of a YFP signal in these two cellular compartments. The co-expression of SAP11_CaPm and MdNYC1 did not show any YFP signal. The RFP signal (depicted in magenta) indicates a successful transformation of protoplasts with the BiFC vector. Chl (depicted in blue) shows the autofluorescence of chlorophyll within the chloroplasts. Microscopic analysis was performed with a Zeiss LSM 800 confocal microscope. Bars represent 20 μm for all micrographs.

Taken together, these results show that SAP11_CaPM interacts with MdTCP16 in addition to the previously shown interaction with MdTCP24 and MdTCP25 [8].

Expression of TCPs in Malus × domestica during infection

It was unclear how an infection with ’Ca. P. mali’ affects the expression of the three TCP-encoding genes being the targets of its effector SAP11_CaPm. Thus, to analyze the expression of MdTCP16, MdTCP24 and MdTCP25 during infection in Malus × domestica host plant, qPCR assays were used to determine expression levels of the respective TCPs and the effector protein SAP11_CaPm (Fig 3).

Fig 3 — *MdTCP* expression in spring and autumn of non-infected (green) and naturally infected (black) apple leaves (A) or graft inoculated and grown in greenhouse plants (B). Naturally infected samples (A) comprise one leaf pool from non-infected trees, and three leaf pools from infected trees. Graft inoculated greenhouse plants (B) comprise leaf samples from 5 non-infected and 5 infected trees, each represented by a data point. Correlation of *MdTCP* and *SAP11*_CaPm expression (C, D, E) in infected *Malus* × *domestica* leaf-samples from spring (grey, n = 8) and autumn (black, n = 8). Lines in graphs C, D, E and F show linear regression of the respective samples either from the greenhouse (dashed line) or from the foil tunnel (solid line). Samples in C, D and E were grouped regarding their growing conditions and plotted to different y-axis due to the differences in the concentration ranges of the different sample subsets. Correlation of *MdTCP*, *SAP11*_CaPm expression and phytoplasma levels (F) in graft-inoculated *Malus* × *domestica* leaf-samples from spring (grey, n = 3) and autumn (black, n = 4). Statistical analysis was performed with multiple t-test. Statistical differences were determined using the Holm-Sidak method, with alpha = 0.05 and linear regression analysis, using GraphPad Prism 7.05 (GraphPad Software Inc.). For pools comprising only one biological replicate, technical replicates accounted to the statistical analysis. Significant differences between groups are indicated with asterisks (* P≤0.05, ** P≤0.01, *** P≤0.001). Data points with very similar values might overlap in the graph and can thus misleadingly appear as a single data point. In only seven out of the ten samples from the greenhouse phytoplasma concentration could be determined (F). This was due to a lack of sample material for the DNA preparation which is necessary for phytoplasma detection.

MdTCP24 was stably expressed throughout the season and its transcript levels did not change upon infection, regardless of whether the trees were grown in the field or in the greenhouse (Fig 3A and 3B). Also, MdTCP25 in leaves from greenhouse plants was stably expressed throughout the season, but in naturally infected samples the expression was significantly higher in samples taken in spring than in those taken later in the season, for both non-infected and infected trees. Furthermore, regarding field-grown trees in spring, MdTCP25 expression is significantly lower in naturally infected samples compared to non-infected samples (Fig 3A and 3B). The expression of MdTCP16 was lower than those of MdTCP24 and MdTCP25. MdTCP16 transcript levels were low in both infected and non-infected samples from spring and higher in samples from autumn. Furthermore, it was tendentially, but not significantly higher in infected autumn samples than in non-infected autumn samples (Fig 3A and 3B). SAP11_CaPm expression could not be detected in naturally infected spring samples, but it was detectable in the leaves from greenhouse samples throughout the season (Fig 3C–3E). SAP11_CaPm expression in leaves tends to be increased when phytoplasma level in the same tissue is high (Fig 3F).

The expression of MdTCP16 significantly correlated positively with the expression of SAP11_CaPm in leaves from trees of the greenhouse (R² = 0.40) and there was a trend of this finding also in leaves from trees cultivated in the foil tunnel (R² = 0.52). MdTCP16 expression also strongly correlated positively (R² = 0.97) with the phytoplasma quantity in leaf samples (Fig 3C and 3F, Table 1). MdTCP16 expression furthermore negatively correlates (R² = 0.74) with the phytoplasma concentration (3F, Table 1). This is opposite to the findings for MdTCP25, whose expression correlated negatively with SAP11_CaPm quantity in leaf samples of trees from the greenhouse (R² = 0.15) and from the foil tunnel (R² = 0.78) (Fig 3D). The negative correlation between MdTCP25 and SAP11_CaPm expression was significant in leaves from the foil tunnel, whereas in the samples from greenhouse tree a trend of a negative correlation was observed (Fig 3D). MdTCP24, in contrast, did neither correlate with the expression of SAP11_CaPm nor with the phytoplasma quantity (Fig 3E and 3F).

Table 1. Linear regression analysis of TCP expression depending on phytoplasma quantity or SAP11_CaPm expression.

95% confidence interval (CI), goodness of fit and significance level (alpha = 0.05) of linear regression, calculated with GraphPad Prism 7.05 (GraphPad Software Inc.), between normalized phytoplasma quantity and SAP11_CaPm, MdTCP16, MdTCP25 and MdTCP24 expression (left panel) and between normalized SAP11_CaPm expression and MdTCPs expression in leaves from greenhouse (middle panel) and from foil-tunnel, respectively (right panel).

	normalized phytoplasma quantity (greenhouse)				normalized SAP11_CaPm expression (greenhouse)			normalized SAP11_CaPm expression (foil tunnel)
	SAP11 _CaPm	MdTCP16	MdTCP25	MdTCP24	MdTCP16	MdTCP25	MdTCP24	MdTCP16	MdTCP25	MdTCP24
95% CI
Slope	-0.020 to 0.358	6.195 to 9.67	-0.096 to -0.019	-0.196 to 0.006	0.194 to 45.46	-0.405 to 0.13	-0.603 to 0.452	-0.912 to 6.262	-0.658 to -0.0958	-0.320 to 0.562
Y-intercept	0.143 to 1.439	-5.168 to 6.743	0.955 to 1.22	0.903 to 1.595	-38.49 to 18.16	0.762 to 1.43	0.435 to 1.75	-2.0 to 3.99	0.907 to 1.38	0.535 to 1.27
X-intercept	-infinity to -0.498	-0.995 to 0.585	12.01 to 54.6	7.031 to +infinity	-78.23 to 1.014	3.4 to +infinity	2.708 to +infinity	-infinity to 0.456	1.923 to 10.28	-infinity to -1.071

Goodness of Fit
R²	0.513	0.965	0.743	0.540	0.403	0.150	0.0135	0.517	0.776	0.127

P value	0.07	<0.001	0.01	0.06	0.049	0.269	0.750	0.107	0.02	0.489
Deviation from zero?	Not Significant	Significant	Significant	Not Significant	Significant	Not Significant	Not Significant	Not Significant	Significant	Not Significant

Equation	Y = 0.169*X + 0.791	Y = 7.932*X + 0.788	Y = -0.057*X + 1.087	Y = -0.095*X + 1.249	Y = 22.83*X-10.16	Y = -0.1377*X + 1.097	Y = -0.07555*X + 1.094	Y = 2.675*X + 0.9928	Y = -0.3771*X + 1.141	Y = 0.1209*X + 0.9024

Number of X values	7	7	7	7	10	10	10	16	16	16

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Discussion

It is known that SAP11_AYWB from AYWB-phytoplasma interacts and destabilizes AtTCP18 in Arabidopsis thaliana [12], but it was so far unknown if SAP11_CaPm from ’Ca. P. mali’ can interact with MdTCP16, the orthologous of AtTCP18 in Malus × domestica, the actual host plant of this phytoplasma species. It has been shown previously that SAP11_CaPm from strain PM19, that shares 99.2% sequence identity to SAP11_CaPm from strain STAA used in this study, interacts with AtTCP18 in a Y2H test [16]. However, to understand the potential effects of the effector protein SAP11_CaPm, it is important to unravel the targets not only in model plants but in the natural host plant and to understand the induced changes in TCP expression in the native pathosystem.

Our data show unequivocally that SAP11_CaPm interacts with MdTCP16, both yeast and in planta, whereas another candidate (MdNYC1) does not interact (Figs 1 and 2). SAP11_CaPm also binds to MdTCP25 and MdTCP24 from Malus × domestica and degrades different AtTCPs at the protein level, among them the orthologue of MdTCP25, i.e. AtTCP4 [16], but it is not known, whether binding of SAP11_CaPm affects the stability of MdTCP16, since it was not able to destabilize the Arabidopsis thaliana orthologue AtTCP18 [11,31]. Interestingly, our study indicates that MdTCP16 expression is induced during phytoplasma infection and might then be counter-regulated by SAP11_CaPm at the protein level. A degradation of MdTCP16 induced by SAP11_CaPm at the protein level could induce MdTCP16 expression on the transcriptional level via a gene-product mediated feedback loop regulation [42]. The MdTCP16 protein might act like a repressor and negatively affects expression of the MdTCP16 gene, meaning that skimming the proteinaceous gene product leads to an increase of its gene expression. That might explain why MdTCP16 transcriptional expression levels are by trend increased in infected plants and that phytoplasma level in leaves is positively correlated with the expression of this transcription factor (Fig 3A–3E) which has been shown also for ‘Ca. P. ziziphi’ infected Ziziphus jujube, another phytoplasma pathosystem where the ZjTCP7 that encodes a MdTCP16 orthologue of Z. jujube, is upregulated in leaves [43]. It is worth noting that–in contrast to MdTCP16 –MdTCP25 expression is adversely i.e., negatively and MdTCP24 expression not at all correlated with SAP11_CaPm expression during infection. This implies that other regulatory mechanisms might be involved in the regulation of MdTCP25 and MdTCP24 expression.

Whatever pathway is affected during MdTCP16 expression, it seems reasonable to speculate that it is important for the phytoplasma to attack MdTCP16 in its plant host.

The increased MdTCP16 expression in autumn samples might be an indication that this TCP is involved in the seasonal control of branching. MdTCP16 is mainly expressed in axillary and flower buds and only weakly in leaves, stems and shoot tips of apple trees [44]. Short photoperiods lead to the expression of BRC1 –an orthologue of MdTCP16 –controlling a complex network that regulates bud dormancy [45]. Interestingly, early bud break is a symptom of Phytoplasma-infected apple trees. It is tempting to speculate that ‘Ca. P. mali’ infection affects MdTCP16 expression via SAP11_CaPm in axillary buds and is thus involved in the induction of early bud break. Our study was, however, focused on gene expression analyses in leaves, thus based on the current data availability it would be very speculative to hypothesize about SAP11_CaPm’s function in other plant tissues.

No SAP11_CaPm expression was detected in leaf samples from naturally infected trees in spring, but the effector was detectable in leaves from greenhouse trees already in spring. This discrepancy might be explained by differences in environmental and physiological circumstances between the fully grown naturally infected trees and the small and young trees from the greenhouse. It is possible that the phytoplasma colonization of the canopy occurs earlier or that the bacterial colonization is more uniform in the foliage part of the smaller greenhouse plants.

’Ca. P. mali’ infection leads to increased soluble sugar content in phloem sap [46] and in leaves [47]. Sugar promotes the bud outgrowth by acting as a repression-signal for BRC1 expression [25,48]. This is neither in line with the increased MdTCP16 expression that we find in phytoplasma infected apple leaves, nor with what has been described for ZjTCP7 expression in phytoplasma infected Chinese jujube [43]. Thus, other factors might outcompete the repressing effect of increased sugar levels on MdTCP16 expression, such as an increased auxin level in leaves of infected apple trees [49]. Auxin induces the MdTCP16 expression but blocks the axillary bud outgrowth in non-infected plants [50,51].

Taken together, BRC1 and its homologues seem to be important molecular targets of SAP11-like effector proteins from different phytoplasma species. The results of this study prove the interaction of SAP11_CaPm and the MdTCP16 transcription factor. SAP11-like proteins might be crucial for the successful phytoplasma colonization of the canopy in spring by downshifting BRC1. In the context of the current knowledge, it can be assumed that this downregulation is involved in the formation of lateral shoot outgrowth and early bud break, which are typical symptoms of ’Ca. P. mali’ infection, and eponymous to the diseases name “apple proliferation” [31]. However, the factors involved in BRC1/MdTCP16/AtTCP18 gene regulation are not easy to detangle, since they seem to be species- and tissue- dependent and regulated in a complex manner. By expressing SAP11-like proteins, phytoplasma target different members of the plant TCP family which serve as molecular hubs, to manipulate their plant hosts in a very sophisticated -but not yet fully understood- manner.

Supporting information

S1 Fig. Reference Sequence of XM_008376500.2.

The sequence includes the CDS for XP_008374722.1 (MdTCP16) with TCP domain and the identified part in the Y2H screen.

(PDF)

Click here for additional data file.^{(303.7KB, pdf)}

S2 Fig. Phytoplasma concentration of leaf and root samples.

The normalized phytoplasma concentration is given as the ratio of the ’Ca. P. mali’ specific 16S gene copies and the Malus x domestica single-copy gene ACO. Phytoplasma concentration was quantified in seven infected leaf samples from greenhouse plants (three from spring and four from autumn), in three naturally infected pooled leaf samples (one from spring, two from autumn) and in one naturally infected pooled root sample (autumn).

(PDF)

Click here for additional data file.^{(221KB, pdf)}

S1 Table. Primers used in this study.

Lowercase letters indicate bases for Gateway-attB site overhangs or SfiI restriction site overhangs.

(PDF)

Click here for additional data file.^{(171.3KB, pdf)}

Acknowledgments

We would like to thank Christine Kerschbamer for lab assistance and Mirko Moser (Fondazione Edmund Mach, San Michele All’Adige, Italy) for discussing the data. Massimiliano Trenti, Erika Corretto from the Free University of Bolzano (Italy) and Pier Luigi Bianchedi from Fondazione Edmund Mach (San Michele all’Adige, Italy) for experimental assistance in grafting and sampling and Cameron Cullinan for English proofreading.

Data Availability

All relevant data are within the article and its Supporting Information files.

Funding Statement

The work of CM and KJ was co-funded by the Autonomous Province of Bozen/Bolzano (Italy, https://www.provinz.bz.it) and the South Tyrolean Apple Consortium (Italy, https://www.apfelwelt.it). The study was realized within the APPLIII and APPLIV project belonging to the Framework agreement in the field of invasive species in fruit growing and major pathologies (PROT. VZL_BZ 09.05.2018 0002552). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS One. doi: 10.1371/journal.pone.0272467.r001

Decision Letter 0

Keith R Davis

19 Sep 2022

PONE-D-22-20188The ‘Candidatus Phytoplasma mali’ effector protein SAP11CaPm interacts with MdTCP16, a class II CYC/TB1 transcription factor that is highly expressed during phytoplasma infection.PLOS ONE

Dear Dr. Janik,

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Reviewer #1: The MS “The ‘Candidatus Phytoplasma mali’ effector protein SAP11CaPm interacts with MdTCP16, a class II CYC/TB1 transcription factor that is highly expressed during phytoplasma infection.” (PONE-D-22-20188) is a very nice little story which deals with an interesting mechanism of effector protein interacting with transcription factors.

The MS is written very well and is also well structured. Nevertheless, I have some critical points which should be considered for revision. The M+M part have to be improved as I could not found the protocol for protoplastation and also the growth conditions of the apple trees (field and greenhouse) are missing. Which apple cultivars are used and did the authors used the same cultivar for grafting (rootstock and scion)? Which Ca. P mali accession did the authors used in the grafted trees? (virulent or avirulent?) Can the interaction of SAP11 and MdTCP16 be different if the tree is infected with a virulent or avirulent accession? What about the phytoplasma titre of the trees, should be shown as suppl. table. Fig. 2: YFP signal seems also be in the membrane. That this is not the case should be checked by colocalization.

Reviewer #2: In this study, Mittelberger et al. investigate the interactions of the Candidatus phytoplasma mali effector protein SAP11 with two host proteins: the transcription factor MdTCP16 and NYC1, a chloroplast reductase. The paper also analyses the expression of three different Malus domestica transcription factors (MdTCP16, MdTCP24 and MdTCP25) during phytoplasma infection over the plant vegetative season. The main focus of the study is improving the knowledge about SAP11 role in Ca. P. mali infection identifying new interactors and describing the expression dynamic of SAP11 and its host targets between spring and autumn.

The manuscript is generally well-written and easy to follow. The two approaches adopted to study SAP11 targets congruently identify MdTCP16 as a new interactor of the effector protein. However, some issues with the methods and analysis of the gene expression studies need to be addressed.

Below are more specific comments by section:

Title, Abstract and Introduction:

- The title appropriately summarizes the content and results of the paper.

- The abstract is detailed, concise, and appropriately condenses the manuscript content.

- The introduction provides enough information to understand the background and the relevance of the study.

Methods:

- A description of the different plant growing conditions would help understand and interpret the results. Since BRC1 are influenced by photoperiod and temperature fluctuations, it is important to know if the plants in greenhouse were maintained in controlled conditions or not. I suggest introducing one or more sections to describe the plant materials and the plant growth conditions in the greenhouse (light, temperature). The sampling strategies, and the sampling size for naturally infected and greenhouse plants need also to be provided to better interpret the results and statistical analysis. See also comment to lines 221-224.

Results

- Line 193: NYC1 and TCP16 do not share common features, do not belong to the same protein family and are localized in different subcellular compartments. The co-expression of SAP11 and MdNYC1 further confirms the lack of interaction between these two proteins already suggested by the Y2H experiment. Therefore, I would not consider this result as the negative control but part of the experiment, since the BiFC experiment was conducted to validate the results obtained by the Y2H. Moreover,

- Line 221-224: the discrepancy mentioned in line 223 seems to refer to greenhouse vs naturally infected plants. Nevertheless, the authors suggested that the discrepancy “might be due to the season-dependent phytoplasma colonization”. This statement needs to be detailed and moved to the Discussion paragraph.

- Figure 3 Panel A and B. The plots show no difference in MdTCP16 expression between healthy and infected plants in spring and autumn. The fact that MdTCP16 expression increases during the season, such as the phytoplasma titer (panel D), is not enough to prove a direct correlation between the two phenomena. Moreover, the fact that MdTCP16 has the same trend of expression in healthy and infected plants shows that the increase of MdTCP16 transcription is correlated to environmental or plant physiological conditions rather than phytoplasma infection.

Panels C and D. It is unclear what samples are plotted here. The size of the data set differs between the two panels. It would be important to report and describe the data sets used to build the plots.

- Lines 251-252 See comment to Figure 3.

Discussion

- Line 286-290 The authors suggest that TCP16 might play a role in the early bud break during the spring. Nevertheless, in springtime TCP16 expression level is the lowest registered during the vegetative season. Moreover, in spring, the expression level of SAP11 was not detectable in the open field and was extremely low in the greenhouse.

A further concern is that the expression analysis of all the genes involved in the study was conducted on leaf samples, but the MdTCP16 is mainly expressed in buds, where it is supposed to play a crucial role. Similarly, the expression of SAP11 in bud cells was not investigated. Consequently, the hypothesis of the involvement of SAP11-TCP16 interaction in the induction of early bud break of phytoplasma infected trees is speculative and not supported by the data presented.

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Reviewer #1: Yes: Alexandra C. U. Furch

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PLoS One. 2022 Dec 15;17(12):e0272467. doi: 10.1371/journal.pone.0272467.r002

Author response to Decision Letter 0

6 Oct 2022

Reviewer #1:

The MS “The ‘Candidatus Phytoplasma mali’ effector protein SAP11CaPm interacts with MdTCP16, a class II CYC/TB1 transcription factor that is highly expressed during phytoplasma infection.” (PONE-D-22-20188) is a very nice little story which deals with an interesting mechanism of effector protein interacting with transcription factors. The MS is written very well and is also well structured. Nevertheless, I have some critical points which should be considered for revision.

The M+M part have to be improved as I could not found the protocol for protoplastation and also the growth conditions of the apple trees (field and greenhouse) are missing.

>>> Comment R1.1.: We introduced a Plant Material subheading in the M+M section, for describing the plant material. Additionally, the procedure for protoplast extraction and transformation is now described in detail.

Which apple cultivars are used and did the authors used the same cultivar for grafting (rootstock and scion)?

>>> Comment R1.2.: The same cultivar was used. The details are now described in the newly added Plant Material subheading of the M+M section.

Which Ca. P mali accession did the authors used in the grafted trees? (virulent or avirulent?)

>>> Comment R1.3.: The trees in the greenhouse have been grafted with the virulent strain PM6, an Italian AT2-subtype isolate derived from a naturally infected tree of cv. Golden Delicious characterized in Bisognin et al. 2007. This information has been added to the respective part in the M+M section. The other trees are infected with a local AT2 strain as determined by Sanger sequencing. Since these trees have not been infected with a pre-cultured pure phytoplasma strain it cannot be ruled out that a mixed infection with other strains is present in these trees. However, this situation resembles the actual one in in the field.

Can the interaction of SAP11 and MdTCP16 be different if the tree is infected with a virulent or avirulent accession?

>>> Comment R1.4.: This is an interesting question. In a previous analysis, variants of SAP11 from different AP strains in South Tyrol have been analyzed (Janik et al. 2017). No SAP11 variants on amino acid level could be identified in this study, leading to the conclusion that SAP11 is highly conserved in 'Ca. P. mali'. To our knowledge there is no report of 'Ca. P. mali' strains that have an aberrant or dysfunctional SAP11 protein or are completely lacking this protein. The role of SAP11 in avirulent and virulent 'Ca. P. mali' strains has not been analyzed, yet. Based on our data we cannot deduce a hypothesis whether SAP11 and MdTCP16 interaction is differing in virulent and avirulent 'Ca. P. mali' strains. It would be indeed interesting to further analyze the function of this effector in this direction in a future study.

What about the phytoplasma titre of the trees, should be shown as suppl. table.

>>> Comment R1.5.: A supplementary table showing the phytoplasma quantification in the roots of the trees from the foil-tunnel used in this study has been added as Table S2. Unfortunately, a phytoplasma quantification in the roots of the small greenhouse trees for each time point has not been performed in parallel to the leaf analysis. However, data about the phytoplasma concentration in leaves is shown in Fig 3D.

Fig. 2: YFP signal seems also be in the membrane. That this is not the case should be checked by colocalization.

>>> Comment R1.6.: We apologize if the pictures that we provided are not fully unambiguous. A cytoplasm localization can sometimes appear as a localization close to the membrane, especially if it is very faint and the enlargement of cells is not high. Comparing the signals coming from YFP and RFP, it is visible that both perfectly co-localize. Free RFP of the transformation control is cytosolic and – due to its small size – also translocated to the nucleus. The MdTCP16-SAP11CaPm-complex (YFP) is also nuclear and cytoplasmic localized and we could not find any indication for a membrane localization of it. To avoid a potential misunderstanding for the readers of the article we increased the contrast in the image to improve the visual subcellular localization.

________________________________________

Reviewer #2:

In this study, Mittelberger et al. investigate the interactions of the Candidatus phytoplasma mali effector protein SAP11 with two host proteins: the transcription factor MdTCP16 and NYC1, a chloroplast reductase. The paper also analyses the expression of three different Malus domestica transcription factors (MdTCP16, MdTCP24 and MdTCP25) during phytoplasma infection over the plant vegetative season. The main focus of the study is improving the knowledge about SAP11 role in Ca. P. mali infection identifying new interactors and describing the expression dynamic of SAP11 and its host targets between spring and autumn.

need to be addressed.

Below are more specific comments by section:

Title, Abstract and Introduction:

-The title appropriately summarizes the content and results of the paper.

-The abstract is detailed, concise, and appropriately condenses the manuscript content.

-The introduction provides enough information to understand the background and the relevance of the study.

Methods:

>>> Comment R2.1.: A new subheading was now introduced to the M+M section for description of plant growth conditions. Additionally, the sampling strategies and sampling size were completed with all information.

Results

>>> Comment R2.2.: The wording was changed accordingly and the BiFC experiment considered as a confirmation of the Y2H results.

Moreover,

>>> Comment R2.3.: The statement was moved to the discussion section and is now explained in more detail.

- Lines 251-252 See comment to Figure 3.

>>> Comment R2.4.: Figure 3 and its legend were changed according to the reviewer’s suggestion. The number of samples is now described in detail in the legend. You are right, that in Panel B there is no significant difference between MdTCP16 expression in non-infected and infected leaves. The rationale for the conclusion that MdTCP16 expression correlates with phytoplasma presence is based on the data depicted in panel D of this figure, in which a strong correlation between MdTCP16 and phytoplasma concentration can be observed. In panel A and B of this figure there is a trend that MdTCP16 is more expressed in infected leaves in autumn, especially in the (smaller) greenhouse trees. However, the difference is neither significant in the group of the greenhouse trees nor in the naturally infected trees. This initial observation (i.e. that there is a trend in autumn) and the fact that phytoplasma concentration in leaves is the highest in leaves in autumn led us to the question if there is a positive correlation between MdTCP16 expression and phytoplasma concentration in the leaves. The results of the correlation analysis are shown in panel D, where samples from greenhouse and naturally infected trees were analyzed together. In this evaluation a positive correlation between phytoplasma concentration and MdTCP16 expression could be observed.

Discussion

>>> Comment R2.5.: We agree and changed the discussion part accordingly and reduced the speculative part regarding an involvement of SAP11 in MdTCP16-mediated induction of early bud-break.

________________________________________

Attachment

Submitted filename: Response to Reviewers.pdf

Click here for additional data file.^{(208.7KB, pdf)}

PLoS One. doi: 10.1371/journal.pone.0272467.r003

Decision Letter 1

Keith R Davis

25 Oct 2022

PONE-D-22-20188R1

The ‘Candidatus Phytoplasma mali’ effector protein SAP11CaPm interacts with MdTCP16, a class II CYC/TB1 transcription factor that is highly expressed during phytoplasma infection.

PLOS ONE

Dear Dr. Janik,

Please submit your revised manuscript by Dec 09 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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Additional Editor Comments:

Although you did a nice job of addressing many of the reviewers' concerns, Reviewer 2 still has some legitimate questions about the data analysis presented in Figure 3. I agree that combining data from treatments as currently presented is questionable and needs to be addressed. Once this is adequately done, the manuscript can be recommended for publication.

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Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Reviewer #1: Dear authors,

Thank you very much for the revised version. All my raised comments were adequately addressed.

Good luck for your future work.

Reviewer #2: The authors have clarified most of the issues I raised during my last review. I still have a few points about Figure 3 that need to be addressed:

Figure 3C: Since the samples plotted here come from different growing conditions (naturally vs graft infected and greenhouse vs foil tunnel) and, as shown in panels A and B, have different expression profiles, they shouldn't be analyzed as a single set of samples. The expression level of MdTCP16 in grafted plants is more than ten times lower than the transcription level in naturally infected trees. Similarly, the correlation analysis should be performed separately.

Figure 3D: Five grafted inoculated samples were plotted in figure 3B, but in figure 3D only three samples for spring and four for autumn were plotted. What is the rationale behind the exclusion of some of the samples?

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Reviewer #1: No

Reviewer #2: No

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PLoS One. 2022 Dec 15;17(12):e0272467. doi: 10.1371/journal.pone.0272467.r004

Author response to Decision Letter 1

2 Nov 2022

Subject: Resubmission of PONE-D-22-20188R1

thank you very much for the valuable input and the comments regarding the previous version of our manuscript with the title “The 'Candidatus Phytoplasma mali' effector protein SAP11CaPm interacts with MdTCP16, a class II CYC/TB1 transcription factor that is highly expressed during phytoplasma infection”. Along the revised manuscript we provide point-by-point responses to every reviewer’s concern. Every author’s response to a reviewer’s comment is indicated with arrows (>>>). All changes that have been performed to the previous version of the manuscript are indicated by tracked changes. The manuscript text, figures, legends, or any other part of the previously submitted documents have not undergone any substantial changes if not requested by the reviewers.

We hope that our manuscript now fulfills all criteria for publication in PLOS ONE.

Reviewer #1: Dear authors,

Thank you very much for the revised version. All my raised comments were adequately addressed.

Good luck for your future work.

Reviewer #2: The authors have clarified most of the issues I raised during my last review. I still have a few points about Figure 3 that need to be addressed:

>>> The correlation analyses shown in the former Fig 3C and Table 1 were changed according to the reviewer’s suggestions. This improved the comprehensibility of the whole graph and thus the interpretation of the results. Expression levels were compared separately and regarding the growing conditions. The statistical correlation analysis was also performed on the separated sample sets as suggested. The adjusted analytical approach did not affect the overall results, i.e. the same results or trends were found in the Figures 3 C-D as in the former Fig 3C. The text in the manuscript was changed according to the changes in the analyses and the graph.

>>> For the phytoplasma quantification in Fig 3D DNA was required whereas for the expression analyses RNA/cDNA was used. Since the phytoplasma concentration was determined in a second moment there was not enough material for a DNA extraction for every sample left. We tried a phytoplasma quantification based on cDNA but did not succeed. The comparison was performed as follows: In a pilot-study with a subset of samples we compared the phytoplasma quantification results using cDNA with those using genomic DNA from the same sample material. Summarizing the results that we got, we found that DNA-based quantification results did not correspond to those based on cDNA performed in parallel. We must admit that we did not further invest time in finding an appropriate phytoplasmal housekeeping gene that could have been used for accurate cDNA-based phytoplasma quantification. That’s why we -unfortunately- cannot show results all ten samples in Fig 3D. To avoid appearing untransparent in this regard, we now mention the reason for the discrepancy in greenhouse sample numbers between 3B and 3F (before 3D) in the figure legend.

Attachment

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Click here for additional data file.^{(67.6KB, pdf)}

PLoS One. doi: 10.1371/journal.pone.0272467.r005

Decision Letter 2

Keith R Davis

14 Nov 2022

The ‘Candidatus Phytoplasma mali’ effector protein SAP11CaPm interacts with MdTCP16, a class II CYC/TB1 transcription factor that is highly expressed during phytoplasma infection.

PONE-D-22-20188R2

Dear Dr. Janik,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Additional Editor Comments (optional):

Thank you for your patience as we worked our way through the reviews. I hope you agree that the manuscript is improved and more useful for the research community. Best of luck in your future studies.

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #2: Yes

**********

6. Review Comments to the Author

Reviewer #2: The authors did a good job and addressed all my concerns.

Only one little comment below:

Lines 272-274: "Lines in graphs C, D, E and F show linear regression of the respective samples either from the greenhouse (dashed line) or from the foil tunnel (solid line)": In Figures 3C-E, the regression lines are all solid and dashed lines are missing

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Reviewer #2: No

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PLoS One. doi: 10.1371/journal.pone.0272467.r006

Acceptance letter

Keith R Davis

7 Dec 2022

PONE-D-22-20188R2

The ‘Candidatus Phytoplasma mali’ effector protein SAP11_CaPm interacts with MdTCP16, a class II CYC/TB1 transcription factor that is highly expressed during phytoplasma infection.

Dear Dr. Janik:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig. Reference Sequence of XM_008376500.2.

The sequence includes the CDS for XP_008374722.1 (MdTCP16) with TCP domain and the identified part in the Y2H screen.

(PDF)

Click here for additional data file.^{(303.7KB, pdf)}

S2 Fig. Phytoplasma concentration of leaf and root samples.

(PDF)

Click here for additional data file.^{(221KB, pdf)}

S1 Table. Primers used in this study.

Lowercase letters indicate bases for Gateway-attB site overhangs or SfiI restriction site overhangs.

(PDF)

Click here for additional data file.^{(171.3KB, pdf)}

Attachment

Submitted filename: Response to Reviewers.pdf

Click here for additional data file.^{(208.7KB, pdf)}

Attachment

Submitted filename: Response to reviewers.pdf

Click here for additional data file.^{(67.6KB, pdf)}

Data Availability Statement

All relevant data are within the article and its Supporting Information files.

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PERMALINK

The ‘Candidatus Phytoplasma mali’ effector protein SAP11CaPm interacts with MdTCP16, a class II CYC/TB1 transcription factor that is highly expressed during phytoplasma infection

Cecilia Mittelberger

Bettina Hause

Katrin Janik

Roles

Abstract

Introduction

Materials and methods

Yeast-two-hybrid screen

Bimolecular fluorescence complementation analysis

Plant material

RNA extraction and cDNA synthesis

DNA extraction

qPCR analysis

Data analysis

Results

Yeast-two-hybrid and bimolecular fluorescence complementation analyses

Fig 1. SAP11CaPm interacts with MdTCP16 but not with MdNYC1 in yeast.

Fig 2. SAP11CaPm interacts with MdTCP16 in planta.

Expression of TCPs in Malus × domestica during infection

Fig 3. MdTCP16 expression increases from spring to autumn, is slightly higher in infected samples and strongly correlates to phytoplasma levels in leaves from infected Malus × domestica.

Table 1. Linear regression analysis of TCP expression depending on phytoplasma quantity or SAP11CaPm expression.

Discussion

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Keith R Davis

Roles

Author response to Decision Letter 0

Decision Letter 1

Keith R Davis

Roles

Author response to Decision Letter 1

Decision Letter 2

Keith R Davis

Roles

Acceptance letter

Keith R Davis

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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The ‘Candidatus Phytoplasma mali’ effector protein SAP11_CaPm interacts with MdTCP16, a class II CYC/TB1 transcription factor that is highly expressed during phytoplasma infection

Fig 1. SAP11_CaPm interacts with MdTCP16 but not with MdNYC1 in yeast.

Fig 2. SAP11_CaPm interacts with MdTCP16 in planta.

Table 1. Linear regression analysis of TCP expression depending on phytoplasma quantity or SAP11_CaPm expression.