Phytoplasma SAP11 effector destabilization of TCP transcription factors differentially impact development and defence of Arabidopsis versus maize
Authors:
Pascal Pecher aff001; Gabriele Moro aff001; Maria Cristina Canale aff001; Sylvain Capdevielle aff001; Archana Singh aff001; Allyson MacLean aff001; Akiko Sugio aff001; Chih-Horng Kuo aff003; Joao R. S. Lopes aff002; Saskia A. Hogenhout aff001
Authors place of work:
John Innes Centre, Department of Crop Genetics, Norwich Research Park, Norwich, United Kingdom
aff001; Luiz de Queiroz College of Agriculture, Department of Entomology and Acarology, University of São Paulo, Piracicaba, Brazil
aff002; Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan
aff003
Published in the journal:
Phytoplasma SAP11 effector destabilization of TCP transcription factors differentially impact development and defence of Arabidopsis versus maize. PLoS Pathog 15(9): e32767. doi:10.1371/journal.ppat.1008035
Category:
Research Article
doi:
https://doi.org/10.1371/journal.ppat.1008035
Summary
Phytoplasmas are insect-transmitted bacterial pathogens that colonize a wide range of plant species, including vegetable and cereal crops, and herbaceous and woody ornamentals. Phytoplasma-infected plants often show dramatic symptoms, including proliferation of shoots (witch’s brooms), changes in leaf shapes and production of green sterile flowers (phyllody). Aster Yellows phytoplasma Witches’ Broom (AY-WB) infects dicots and its effector, secreted AYWB protein 11 (SAP11), was shown to be responsible for the induction of shoot proliferation and leaf shape changes of plants. SAP11 acts by destabilizing TEOSINTE BRANCHED 1-CYCLOIDEA-PROLIFERATING CELL FACTOR (TCP) transcription factors, particularly the class II TCPs of the CYCLOIDEA/TEOSINTE BRANCHED 1 (CYC/TB1) and CINCINNATA (CIN)-TCP clades. SAP11 homologs are also present in phytoplasmas that cause economic yield losses in monocot crops, such as maize, wheat and coconut. Here we show that a SAP11 homolog of Maize Bushy Stunt Phytoplasma (MBSP), which has a range primarily restricted to maize, destabilizes specifically TB1/CYC TCPs. SAP11MBSP and SAP11AYWB both induce axillary branching and SAP11AYWB also alters leaf development of Arabidopsis thaliana and maize. However, only in maize, SAP11MBSP prevents female inflorescence development, phenocopying maize tb1 lines, whereas SAP11AYWB prevents male inflorescence development and induces feminization of tassels. SAP11AYWB promotes fecundity of the AY-WB leafhopper vector on A. thaliana and modulates the expression of A. thaliana leaf defence response genes that are induced by this leafhopper, in contrast to SAP11MBSP. Neither of the SAP11 effectors promote fecundity of AY-WB and MBSP leafhopper vectors on maize. These data provide evidence that class II TCPs have overlapping but also distinct roles in regulating development and defence in a dicot and a monocot plant species that is likely to shape SAP11 effector evolution depending on the phytoplasma host range.
Keywords:
Maize – Sequence alignment – Transcription factors – Genetically modified plants – Leaves – Flowering plants – Arabidopsis thaliana – Phytoplasmas
Introduction
Phytoplasmas (“Candidatus (Ca.) Phytoplasma”) are economically important plant pathogens that infect a broad range of plant species. The more than 1000 phytoplasmas described so far comprise three distinct clades within a monophyletic group of the class Mollicutes that are characterized by the lack of a bacterial cell wall and small genomes (580 kb to 2200 kb) [1–3]. These fastidious pathogens are restricted to the phloem sieve cells of the plant vasculature and depend on phloem-sap-feeding insect vectors, including leafhoppers, planthoppers and psyllids, for transmission and spread in nature [4]. Many phytoplasmas induce dramatic changes in plant architecture such as increased axillary branching (often referred to as witches’ broom), formation of leaf-like flowers (phyllody), the production of green floral organs such as petals and stamens (virescence), changes of leaf shape, and premature bolting [5–10].
Phytoplasmas change plant architecture via the secretion of proteinaceous effectors that interact with and destabilize plant transcription factors with fundamental roles in regulating plant development. Effectors of Aster yellows phytoplasma strain Witches Broom (AY-WB; “Ca. Phytoplasma asteris”) are particularly well characterized. AY-WB and its predominant leafhopper vector Macrosteles quadrilineatus have broad host ranges that are mostly dicots, including Arabidopsis thaliana [6]. SAP11 destabilizes Arabidopsis TEOSINTE BRANCHED1-CYCLOIDEA-PROLIFERATING CELL FACTOR (TCP) transcription factors, and specifically class II TCPs, leading to the induction of axillary branching and changes in leaf shape of this plant [8,11], and SAP54 degrades Arabidopsis MADS-box transcription factors leading to changes in flower development that resemble phyllody and virescence symptoms [9,12]. Moreover, both effectors modulate plant defence responses leading to increased colonization of M. quadrilineatus on A. thaliana [8,9,13]. For SAP11AYWB this involves the inhibition of jasmonate (JA) synthesis [8]. SAP11 and SAP54 homologs of other phytoplasmas also target TCPs and MADS, respectively, leading to corresponding changes in plant development and architecture [10,14–16]. The majority of phytoplasma effector genes lie within composite-transposon-like pathogenicity islands named potential mobile units (PMUs) that are prone to recombination and horizontal gene transfer [17–20].
Maize bushy stunt phytoplasma (MBSP) belongs to the Aster yellows (AY) group (16SrI) “Ca. P. asteris” [21] and is the only known member of this group to be largely restricted to maize (Z. mays L.), whereas the majority, including AY-WB, are transmitted by polyphagous insects and infect dicotyledonous plants [13,22]. MBSP is transmitted by the maize-specialist insects Dalbulus maidis and D. elimatus; both MBSP and insect vectors are thought to have co-evolved with maize since its domestication from teosinte [23]. Symptoms of MBSP-infected maize plants include the formation of long lateral branches, decline in ear development and emergence of leaves that are often twisted with ripped edges and that display chlorosis and reddening [24]. We previously identified a SAP11 homolog in the MBSP genome [22] and SAP11MBSP is identical in sequence among multiple MBSP isolates collected from Mexico and Brazil [24]. SAP11AYWB and SAP11MBSP lie on microsyntenic regions within the phytoplasma genomes, indicating that these effectors are likely to have common ancestry [22]. However, D. maidis does not produce more progeny on MBSP-infected plants that show advanced disease symptoms; the insects prefer infected plants that are non-symptomatic [25]. In this study we wished to compare the roles of SAP11AYWB and SAP11MBSP in symptom induction and plant defence to insect vectors of A. thaliana and maize.
TCP transcription factors comprise an ancient plant-specific family [26] that are distinguished from other transcription factors by a conserved ± 60 amino acid TCP domain [27]. The TCP domain consists of a helix-loop-helix region that form TCP homo or heterodimers and a basic region that mediates interactions of TCP dimers with DNA motifs [28] and is required for SAP11 binding to TCPs [11]. TCP transcription factors are grouped into three clades based on TCP domain sequences: (i) class I PROLIFERATING CELL FACTOR-type TCPs (PCF clade); (ii) class II CINCINNATA-type TCPs (CIN clade); and (iii) class II CYCLOIDEA/TEOSINTE BRANCHED 1-type TCPs (CYC/TB1-clade) [29]. The latter is also known as the glutamic acid-cysteine-glutamic acid (ECE) clade [30]. PCFs promote cell proliferation, whereas CIN clade TCPs promote leaf and petal cell maturation and differentiation and have antagonistic roles to PCFs [31–34]. The ECE clade includes maize TEOSINTE BRANCHED 1 (TB1) and TB1 homologs of A. thaliana BRANCHED 1 (BRC1 (AtTCP18)) and BRC2 (AtTCP12), that repress the development of axillary branches in plants [35–38], and CYCLOIDEA (CYC) that control flower symmetry [39]. TB1 and genes in the TB1 network have been targeted for selection during maize domestication from a teosinte ancestor [40–41].
Here we show that SAP11AYWB and SAP11MBSP have overlapping but distinct specificities for destabilizing class II TCP transcription factors. The SAP11 effectors induce unique phenotypes in Arabidopsis and maize that indicate divergent roles of class II TCP transcription factors in regulating development and defence in the two plant species. We argue that SAP11MBSP evolution may be constrained due to the specific functionalities of class II TCPs in maize.
Results
Phytoplasma SAP11AYWB binds and destabilizes both Arabidopsis CIN and CYC/TB1 TCPs and SAP11MBSP only CYC/TB1 TCPs
SAP11AYWB and SAP11MBSP interaction specificities for Arabidopsis TCPs (AtTCPs) were investigated via yeast two-hybrid (Y2H) assays and protein destabilization assays in A. thaliana mesophyll protoplasts. In the protoplast experiments, SAP11AYWB destabilized the majority of AtCIN-TCPs and none of the class I AtTCPs tested (Fig 1A, S1 and S2 Figs), confirming previous results [8]. In addition, SAP11AYWB also destabilized CYC/TB1-TCPs BRC1 (AtTCP18) and BRC2 (AtTCP12) (Fig 1A). In contrast, SAP11MBSP destabilized the CYC/TB1 TCPs BRC1 (AtTCP18) and BRC2 (AtTCP12), whereas 7 out of 8 class II AtCIN-TCPs and all tested class I AtTCPs remained stable (Fig 1A). The Y2H assays showed that SAP11AYWB interacts with Arabidopsis CIN-TCPs (Fig 1B), confirming previous data [8,11], whereas SAP11MBSP did not (Fig 1B). However, both SAP11AYWB and SAP11MBSP interacted with CYC/TB1 BRC1 (AtTCP18) and BRC2 (AtTCP12) (Fig 1B). Therefore, SAP11MBSP binds and destabilizes a narrower set of class II TCPs compared to SAP11AYWB.
Alignments of the SAP11AYWB and SAP11MBSP amino acid sequences revealed conservation of the signal peptide and C-terminal sequences, while the central region that includes the domains required for nuclear localization and TCP-binding of SAP11AYWB [11, 17] are more variable (Fig 2A). SAP11AYWB has a bipartite NLS that is required for nuclear localization of this effector [11]. However, the NLS sequence is not conserved in SAP11MBSP; instead NLStradamus [42] predicted the NLS to locate in the C-terminal part of the MBSP effector (Fig 2A). Localization studies with GFP-tagged SAP11 proteins in protoplasts, in the presence of BRC2 (AtTCP12), showed that both SAP11 proteins localize to plant cell nuclei, in contrast to GFP alone, which is distributed throughout the cells (S3 Fig). Therefore, the two SAP11 proteins target cell nuclei in the presence of BRC2 (AtTCP12). We previously demonstrated that the MEILKQKAEEETKNL of SAP11AYWB is required for TCP-binding, whereas deletion of the C-terminal KEEGSSSKQPDDSKK sequence did not affect the TCP binding of the effector [11]. Therefore, we assigned the MEILKQKAEEETKNL sequence as the TCP-binding domain (Fig 2A). To dissect what sequences within the SAP11 protein determine binding specificity to TCPs, we generated chimeras between SAP11AYWB and SAP11MBSP and studied their binding to CYC/TB1 BRC1 (AtTCP18) and CIN TCP2 (Fig 2B, S4A Fig). Therefore, the TCP-interaction domain plays a role in determining SAP11 binding specificity to the two TCPs (Fig 2A and 2B, S4A Fig).
To investigate which region of the TCP domain determine SAP11 binding specificity, chimeras of the basic region and helix loop helix regions of the TCP domains of CIN-TCP AtTCP2 and CYC/TB1-TCP BRC1 (AtTCP18) were constructed (Fig 2C and 2D) and tested for interactions with the two SAP11 proteins. SAP11AYWB and SAP11MBSP interacted with the TCP domains of AtTCP2 and BRC1 (AtTCP18) (Fig 2D, S4B Fig), as observed for full-length TCPs (Fig 1B), confirming that the TCP domain itself is sufficient for SAP11 interaction and specificity. Furthermore, SAP11AYWB interacted with all AtTCP2-BRC1 (AtTCP18) chimeras used in the assay (Fig 2D, S4B Fig), whereas SAP11MBSP interacted with chimeras containing BRC1 (AtTCP18) helix-loop-helix and AtTCP2 basic regions, but not with those composed of AtTCP2 helix-loop-helix and BRC1 (AtTCP18) basic region or with mixed helix, loop and helix sequences (Fig 2D, S4B Fig). Therefore, the entire helix-loop-helix region of the TCP domain is required for the specific binding of SAP11MBSP to CYC/TB1 TCPs. Taken together, multiple amino acids are likely to determine the specificity of SAP11-TCP interactions.
A. thaliana plants stably expressing SAP11MBSP and SAP11AYWB phenocopy brc1 brc2 mutant or CIN-TCP knock down lines
To investigate if the SAP11 binding specificity to TCPs aligns with in planta interactions, phenotypes of A. thaliana Col-0 stable transgenic lines that produce SAP11AYWB and SAP11MBSP under control of the 35S promoter (Fig 1C) were compared to those of the A. thaliana brc1-2 brc2-1 double mutant, hereafter referred to as the brc1 brc2 mutant, which is a null mutant for both CYC/TB1-TCPs BRC1 (AtTCP18) and BRC2 (AtTCP12) [35] and the 35S::miR319a x 35S::miR3TCP line in which CIN-TCPs are knocked down [31]. Whereas the crinkled leaves of 35S::SAP11AYWB lines phenocopied those of 35S::miR319a x 35S::miR3TCP (Fig 1D) [8], leaves of 35S::SAP11MBSP lines were not crinkled and more similar to WT Col-0 leaves (Fig 1D). Rosette diameters of the 35S::SAP11AYWB and 35S::miR319a x 35S::miR3TCP lines were smaller than WT Col-0 plants, unlike the rosettes of 35S::SAP11MBSP and A. thaliana brc1 brc2 mutant lines that looked similar to those of WT plants (Fig 1F). Both 35S::SAP11AYWB and 35S::SAP11MBSP lines produced significantly more primary rosette-leaf branches (RI) [35] than WT plants. With exception of the 35S::SAP11MBSP line 3 that had a lower number of RIs, the production of RI was similar to the A. thaliana brc1 brc2 mutant. In contrast, 35S::miR319a x 35S::miR3TCP plants produced a reduced number of RI compared to WT Col-0 (Fig 1E and 1G, S5E Fig). Therefore, 35S::SAP11MBSP lines phenocopied the A. thaliana brc1 brc2 mutant and the 35S::SAP11AYWB lines both the A. thaliana brc1 brc2 and 35S::miR319a x 35S::miR3TCP mutant lines, indicating that SAP11AYWB destabilizes Arabidopsis CIN and CYC/TB1 TCPs and SAP11MBSP the CYC/TB1-TCPs BRC1 (AtTCP18) and BRC2 (AtTCP12), in agreement with the results of protoplast-based destabilization and Y2H binding assays.
Beyond phenotypes described above, we found that the 35S::miR319a x 35S::miR3TCP and 35S::SAP11AYWB lines produced less rosette leaves compared to WT plants, unlike the A. thaliana brc1 brc2 and 35S::SAP11MBSP lines (S5A Fig). Bolting time, plant height and numbers of primary cauline-leaf branches (CI) [35] were variable among the 35S::SAP11AYWB and 35S::SAP11MBSP lines (S5B–S5E Fig). Roots of 35S::miR319a x 35S::miR3TCP and 35S::SAP11AYWB lines were consistently shorter compared to WT plants as described by Lu et al. [43]. In contrast, the root length of A. thaliana brc1 brc2 and 35S::SAP11MBSP lines did not show obvious differences compared to those of WT plants (S6 Fig).
SAP11AYWB impairs A. thaliana defence responses to M. quadrilineatus in contrast to SAP11MBSP
We previously showed that the AY-WB insect vector M. quadrilineatus produces 20–30% more progeny on 35S::SAP11AYWB A. thaliana [8]. By repeating this experiment and including 35S::SAP11MBSP A. thaliana, we confirmed the previous result for 35S::SAP11AYWB A. thaliana but not for 35S::SAP11MBSP A. thaliana (Fig 3A). Therefore, SAP11AYWB appears to modulate plant defences in response to M. quadrilineatus, whereas SAP11MBSP does not. To test this further, the transcriptomes of wild type, 35S::SAP11AYWB and 35S::SAP11MBSP A. thaliana with and without exposure to M. quadrilineatus were compared via RNA-seq (S1 Table, GEO accession GSE118427). PCA showed that, in samples exposed to M. quadrilineatus, 35S::SAP11MBSP and WT Col-0 group together, whereas the 35S::SAP11AYWB samples form a separate group (Fig 3B). Therefore, SAP11AYWB has a measurable impact on the transcriptome of A. thaliana, unlike SAP11MBSP.
Analyses of differentially expressed genes (DEGs) of Col-0 and transgenic plants exposed to M. quadrilineatus identified 96 DEGs for 35S::SAP11AYWB versus Col-0 and only one DEG for 35S::SAP11MBSP versus Col-0 (Fig 3C and 3D). Hierarchical cluster of the DEGs expression levels was in agreement with the PCA results demonstrating that the M. quadrilineatus-exposed 35S::SAP11AYWB treatments cluster separately from those of Col-0 and 35S::SAP11MBSP (Fig 3E, S2 Table). Moreover, M. quadrilineatus-exposed 35S::SAP11AYWB treatments cluster together with non-exposed samples. Of the 96 DEGs 30 have a role in regulating plant defence responses, including hormone and secondary metabolism, such as Myb, AP2/EREBP and bZIP transcription factors, receptor kinases, cytochrome P450 enzymes, proteases, oxidases and transferases (highlighted in yellow, S3 Table). The 96 genes also included 11 natural anti-sense genes and at least 30 genes with unknown functions. Taken together, these data indicate that defence responses to M. quadrilineatus are suppressed in 35S::SAP11AYWB plants.
Identification of maize TCP transcription factors
To investigate SAP11 interactions with maize TCPs we first identified maize TCP sequences. The CDS of 44 Z. mays (Zm) TCPs available on maize TFome collection [44] were extracted from the Grass Regulatory Information Server (GRASSIUS) (http://grassius.org/grasstfdb.html) [45]. We identified two class II CYC/TB1-TCPs, including TB1 (ZmTCP02) and ZmTCP18, 10 class II CIN-TCPs and 17 class I PCF-like TCPs. The ZmTCPs were assigned to groups based on characteristic TCP domain amino acids conserved in each of the groups, highlighted in yellow, red and green (Fig 4) [29]. In contrast to A. thaliana, maize appears to have an additional group of class II TCPs that share amino acids conserved in the TCP domains of both CIN and TB1/CYC TCPs (Fig 4). One of these is BRANCHED ANGLE DEFECTIVE1 (BAD1), which is expressed in the pulvinus to regulate branch angle emergence of inflorescences, particularly the tassel [46]. BAD1 was placed in a subclade of CYC-TB1 TCPs named as TCP CII. Hence, we assigned all members in this additional group to TCP CII. TCPs similar to TCP CII appear to be absent in the monocots sorghum (S. bicolor) and rice (O. sativa) (S7 and S8 Figs, S4 Table). Seven CIN-TCPs of maize, four CIN-TCPs of rice and five CIN-TCPs of sorghum are potentially regulated by miR319a (Fig 4, S7–S9 Figs). While this study was ongoing, Chai et al. [47] reported the expression characteristics of 29 maize TCPs. To promote consistency, we adopted their nomenclature for these TCPs as ZmTCP01 to ZmTCP29, and continued the numbering of the additional 15 maize TCP genes extracted from GRASSIUS as ZmTCP30 to ZmTCP44 (Fig 4, S4 Table).
Phytoplasma SAP11 homologs interact with and destabilize maize class II TCPs
Y2H assays revealed that SAP11MBSP interacts with the CYC/TB1-TCPs TB1 (ZmTCP02) and ZmTCP18, but not with ZmTCP members of the CIN and CII subgroups (Fig 5A). In contrast, SAP11AYWB interacted also with CIN and CII ZmTCPs (Fig 5A). GFP-SAP11MBSP and GFP-SAP11AYWB destabilized HA-tagged TB1 (ZmTCP02) and ZmTCP18 in maize protoplasts in contrast to GFP controls (Fig 5B, S10 Fig), indicating that the SAP11 homologs also destabilize maize TCPs in maize cells.
Stable SAP11MBSP and SAP11AYWB transgenic maize plants lack female and male sex organs, respectively
SAP11AYWB and SAP11MBSP were cloned as N-terminal 3XFLAG tag fusions downstream of the maize Ubiquitin promoter, and transformed into HiIIAXHiIIB hybrid Z. mays. Ubi::FLAG-SAP11MBSP primary transformants (T0) were female sterile, but produced pollen, which were used for fertilizing flowers of a wild type HiIIA plant. In contrast, Ubi::FLAG-SAP11AYWB primary transformants were male sterile, but produced flowers, which were successfully fertilized with pollen from a HiIIA plant. The T1 progenies of both crosses had similar production of SAP11 proteins (Fig 5C) and were further phenotyped.
Unlike WT HiIIA, Ubi::FLAG-SAP11MBSP T1 plants produced multiple tillers arising from the base of the main culm (Figs 5D (a, c) and 6). Both main culm and tillers produced apical male inflorescences with tassels that carried anthers with pollen (Figs 5D (j, l, insets 7, 10, 11) and 6). These pollen were fertile, as they were used to pollinate HiIIA female inflorescence for seed reproduction. At the upper nodes of the main culm where in WT plants short primary lateral branches with apical ears would develop from the leaf sheath (Figs 5D (g) and 6), long primary lateral branches emerged that also had apical tassels (Figs 5D (i, inset 3) and 6). Hence, Ubi::FLAG-SAP11MBSP plants were female sterile. These phenotypes of Ubi::FLAG-SAP11MBSP plants are similar to those of the Z. mays tb1 mutant (Fig 6) [40,48]. Essentially, Ubi::FLAG-SAP11MBSP and Z. mays tb1 mutant lines resemble teosinte, though the latter produces small ears located at multiple lateral positions of the primary lateral branches (Fig 6) [49]. Therefore, Ubi::FLAG-SAP11MBSP plants phenocopy the maize tb1 mutant, in agreement with the results of yeast two-hybrid and protoplast destabilization assays showing that SAP11MBSP destabilizes CYC/TB1 TCPs.
MBSP-infected maize plants show multiple tillers developing from the base of the main culm and primary lateral branches with apical tassels [24], like Ubi::FLAG-SAP11MBSP and tb1 maize plants (Fig 6). The MBSP-infected maize plants produce ears at the same position where the elongated lateral branches appear, though the ears are fewer in number, substantially smaller and produce less seed than WT non-infected plants. The latter may occur because of tillering, which distributes energy/carbon to the many tillers rather than the development of kernels leading to reduced fertility, and possibly because SAP11MBSP inhibits development of female reproductive organs, like in Ubi::FLAG-SAP11MBSP and tb1 maize plants. In the case of the transgenic plants, SAP11MBSP is being expressed from the start, when the plants grow up, whereas for the MBSP infection, the plants are exposed to the effector later when already partly developed (maize plants are infected with MBSP when they are 3 weeks old, as the leafhopper vectors tend to kill the maize seedlings when they are younger). Phytoplasmas are phloem-limited, and SAP11 and other effectors secreted by the phytoplasmas can unload from the phloem and migrate to distant tissues, including the apical meristem [17,50]. Therefore, it is highly likely that SAP11 interact with and destabilize TCP transcription factors during infection, in agreement with the symptoms of MBSP-infected plants.
Ubi::FLAG-SAP11AYWB T1 plants also produced more tillers from the base of the main culm, but were shorter than WT HiIIA and Ubi::FLAG-SAP11MBSP (Fig 5D (a, b, c)). The majority of leaves of Ubi::FLAG-SAP11AYWB plants had curly edges, unlike Ubi::FLAG-SAP11MBSP and HiIIA plants (Fig 5D (d, e, f, h, inset 2)). Ubi::FLAG-SAP11AYWB plants produced red-coloured silks emerging directly from the leaf sheath without prior ear formation (Figs 5D (h, inset 2) and 6). Upon pollination of the red-coloured silks, ears with reduced husk leaves and exposed corn emerged (Fig 5E (o)). As well, the tip of the main culm and tillers carried tassel-like structures with female flowers and emerging silks (Figs 5D (k, insets 8, 9) and 6). Pollination of these silks with HIIA pollen induced the formation of a few corns (Fig 5E (m,n)). Thus, SAP11AYWB induces tassel feminization and interferes with leaf development, including the modified leaves that generate the husk of the ear.
SAP11AYWB or SAP11MBSP do not alter maize susceptibility to M. quadrilineatus and D. maidis
We investigated if SAP11AYWB and SAP11MBSP modulate maize processes in response to the AY-WB and MBSP insect vectors M. quadrilineatus and D. maidis, respectively. We did not observe any differences in fecundity of both insect vectors on HiIIA, Ubi::FLAG-SAP11AYWB and Ubi::FLAG-SAP11MBSP plants (Fig 7A and 7B). PCA of RNA-seq data from WT and transgenic maize plants indicate that SAP11AYWB and SAP11MBSP modulate maize transcriptomes with SAP11AYWB having a larger effect than SAP11MBSP (Fig 7C and 7D, S5 and S6 Tables, GEO: GSE118427), in agreement with morphological data of the maize lines (Figs 5 and 6). However, M. quadrilineatus-exposed HiIIA Ubi::FLAG-SAP11AYWB and Ubi::FLAG-SAP11MBSP maize clustered together and separately from non-exposed maize in PCA (Fig 7C). D. maidis exposed maize samples grouped with the non-exposed ones (Fig 7D), suggesting that the SAP11 homologs do not have obvious effects on transcriptome responses of maize to the insects. Moreover, M. quadrilineatus has a larger impact and D. maidis a minor impact on maize gene expression (Fig 7C and 7D). Together, these data indicate that SAP11AYWB and SAP11MBSP do not alter maize susceptibility to M. quadrilineatus and D. maidis.
Discussion
We found that SAP11AYWB and SAP11MBSP have overlapping, but distinct, binding specificities for class II TCP transcription factors. We identified the TCP-interaction domain that is involved in determining the specificity of SAP11AYWB and SAP11MBSP binding to CYC/TB1 and CIN-TCPs and found that the two effectors bind to the helix-loop-helix region of the TCP domain of the TCP transcription factors. The helix-loop-helix region of the TCP domain is required for TCP-TCP dimerization and configuration of the TCP domain beta sheets of both TCP transcription factors in a way that allows binding of the beta sheets to promoters [28]. We also found that SAP11-TCP binding specificities are correlated with the ability of the SAP11 homologs to destabilize these TCPs in leaves [8] and protoplasts (this study) and the induction of specific phenotypes in plants [8, this study]. Whereas it remains to be resolved how SAP11 destabilizes TCPs, it is clear that SAP11 is highly effective at destabilizing TCPs in plants as evidenced by the specific SAP11-induced changes in A. thaliana and maize architectures that phenocopy TCP mutants and knock-down lines of these plants.
TCP domains of each TCP (sub)class have characteristic amino acid sequences that have remained conserved after the divergence of monocots and eudicots [51]. The helix-loop-helix regions are characteristic for each TCP (sub)class and are conserved among plants species, including dicots and monocots. We found that SAP11 binding specificity is determined by TCP (sub)class rather than plant species, as SAP11MBSP specifically interacts with class II CYC/TB1-TCPs of both A. thaliana and maize, and not class II CIN-TCP and class I TCPs of these divergent plant species. Similarly, SAP11AYWB interacts with all class II TCPs and not the class I TCPs of A. thaliana and maize. Therefore, SAP11AYWB and SAP11MBSP binding specificity is likely to involve multiple amino acids within the TCP-interaction domain of the SAP11 proteins and the helix-loop-helix region of the TCP domain.
We found that SAP11MBSP interacts with and destabilizes TCPs of the TB1 clade, including A. thaliana BRC1 (AtTCP18) and BRC2 (AtTCP12) and maize TCP02 and TCP18. These binding specificities are supported by plant phenotypes; A. thaliana 35S::SAP11MBSP and maize Ubi::FLAG-SAP11MBSP lines phenocopy A. thaliana brc1 brc2 lines and maize tb1 lines, respectively. The A. thaliana 35S::SAP11MBSP lines show stem proliferations, in agreement with A. thaliana BRC1 (AtTCP18) and BRC2 (AtTCP12) and maize TB1 (ZmTCP02) being suppressors of axillary bud growth [38,52–54]. We also show that A. thaliana 35S::SAP11MBSP and brc1 brc2 lines produce fully fertile flowers, whereas maize Ubi::FLAG-SAP11MBSP plants produced only male tassels and no female inflorescences like maize tb1 plants [40,48]. This is in agreement with BRC1 (AtTCP18) not directly affecting A. thaliana flower architecture [55,56], and maize TB1 (ZmTCP02) being a direct positive regulator of MADS-box transcription factors that control maize female inflorescence architecture [41]. Interestingly, many phytoplasmas have SAP54 effectors, which degrade MADS-box transcription factors leading to the formation of leaf-like sterile flowers [9,10,57,58] whereas no effector gene with sequence similarity to SAP54 was identified in MBSP [24]. It is possible that the maize-specialist phytoplasma strain does not require an additional effector (such as SAP54) to modulate floral development of its host, as SAP11MBSP indirectly targets flowering via TB1 (ZmTCP02).
AtTCP10, which is a CIN-TCP, appears to be destabilized by both SAP11 effectors. This is unexpected given that TCP domains are extremely conserved among TCPs in which those of the CIN-TCPs and CYC/TB1 TCPs are distinct (S11 Fig). We demonstrate that SAP11MBSP binding to CYC/TB1 TCPs requires the entire helix-loop-helix region of CYC/TB1 TCPs, as replacement of the loop or helices with that of a CIN-TCP prevents binding of SAP11MBSP. Based on this, SAP11MBSP is unlikely to bind AtTCP10 directly. TCPs are known to regulate the expression of each other and may also form complexes and, therefore, the expression and abundance of (some) CIN-TCPs may be indirectly affected by deregulation of (SAP11MBSP-mediated) CYC/TB1 TCPs.
Whereas SAP11MBSP interacts and destabilizes TB1 TCPs, SAP11AYWB interacts with all class II TCPs of A. thaliana and maize, in agreement with A. thaliana 35S::SAP11AYWB lines phenocopying both A. thaliana brc1 brc2 and A. thaliana 35S::miR319a x 35S::miR3TCP lines. Information about the role of TCPs in maize development are limited, potentially due to redundant functions of TCPs belonging to the same subgroup and the challenges of obtaining multiple knockdown lines. Therefore, at this time we do not know if maize Ubi::FLAG-SAP11AYWB lines phenocopy maize mutant lines for all CIN and CII TCPs. Nonetheless the leaf crinkling phenotypes of Ubi::FLAG-SAP11AYWB maize plants are in agreement with what is known about the functions of CIN TCPs in Arabidopsis where CIN TCPs play a role in leaf development [8,33,59]. The CII subgroup member BAD1 regulates branch angle emergence of the maize tassel [46] indicating that CII TCPs regulate male inflorescence development in maize. Such a role of these TCPs in maize is consistent with the phenotype of Ubi::FLAG-SAP11AYWB maize plants, given that these produce only female reproductive organs; that is, male developmental organs may not be produced due to absence of CII (and CIN) TCPs in Ubi::FLAG-SAP11AYWB maize plants. Therefore, our finding that Ubi::FLAG-SAP11AYWB maize plants solely producing female inflorescences and no tassels expands the current knowledge about maize CII and CIN-TCPs to a potential role in plant sex determination. We cannot fully exclude the possibility that SAP11AYWB destabilizes other proteins in maize, though we think this is unlikely given our finding that SAP11-TCP interactions are specific involving conserved TCP helix-loop-helix sequences and that SAP11AYWB induces changes in A. thaliana development that are entirely consistent with destabilization of class II TCPs in this plant.
We previously demonstrated that 35S::SAP11AYWB A. thaliana plants are affected in jasmonate production and LOX2 expression upon wounding and that the AY-WB insect vectors produce more progeny on LOX2-silenced plants [8]. A number of TCPs have roles in plant JA production regulation [32,60–65]. Here, we show a clear role of SAP11AYWB suppression of plant defence response genes to M. quadrilineatus, including those involved in phytohormone responses. These genes were not differentially regulated in SAP11MBSP plants response to M. quadrilineatus, indicating that destabilization of CIN-TCPs alone or in combination with Arabidopsis BRC1 (AtTCP18) and BRC2 (AtTCP12) alters plant defence responses to M. quadrilineatus. SAP11AYWB does not promote M. quadrilineatus and D. maidis fecundity on maize suggesting that maize class II TCPs do not play a major role in regulating defence responses of maize leaves. Therefore, class II TCPs appear to regulate plant defence responses in leaves of Arabidopsis but not in maize.
MBSP and the insect vectors D. maidis and D. elimatus are thought to have co-evolved with maize since its domestication from teosinte [23]. We previously sequenced the genomes of MBSP isolates from geographically distant locations and found single nucleotide polymorphisms (SNPs) throughout the genomes of these isolates but that SAP11MBSP remained conserved [24]. The effector may be subject to purifying selection because the destabilization of maize TB1 TCPs and subsequent induction of axillary branching and inhibition of female flower production promote MBSP fitness in maize in a manner that is so far unknown. As well, SAP11MBSP evolution may be constrained by possibly negative effects of maize CIN and ECE TCP destabilization on MBSP fitness or because SAP11MBSP alleles that destabilize other maize TCPs may not be selected in MBSP populations because maize TCPs do not impact D. maidis fitness. Finally, both D. maidis and MBSP predominantly colonize maize, whereas M. quadrilineatus and AYWB colonize a wide range of plants species presenting the possibility that a positive effect of SAP11 on insect fecundity may have more benefit for a generalist phytoplasma and insect vector than for more specialized ones.
In conclusion, we found that SAP11 effectors of AY-WB and MBS phytoplasmas have evolved to target overlapping but distinct class II TCPs of their plant hosts and that these transcription factors also have overlapping but distinct roles in regulating development in these plant species. In addition, TCPs may or may not impact plant defence responses to phytoplasma leafhopper vectors. The distinct roles of TCPs in regulating plant developmental and defence networks are likely to shape SAP11 effector evolution of phytoplasma.
Material and Methods
Generation of Gateway compatible entry clones
We generated Gateway compatible entry clones for all experiments, except for the constructs to transform maize. The cloning of the codon-optimized version of SAP11AYWB without the sequence corresponding to the signal peptide into pDONR207 is described previously [8]. The cloning of sequences corresponding to the open reading frames (ORFs) of AtTCP2, AtTCP3, AtTCP4, AtTCP5, AtTCP7, AtTCP10, AtTCP13 and AtTCP17 (S4 Table) into pDONR207 was also done previously [7]. The full-length ORF of AtTCP6, AtTCP8, AtTCP9, AtTCP12, AtTCP14 and AtTCP18 (S4 Table) were PCR amplified from complementary DNA (cDNA) with gene-specific primers that contain partial sequences of the attB1 and attB2 Gateway recombination sites (S7 Table). The fragments were further amplified with attB1 and attB2 adapter primers and cloned into pDONR207 with Gateway BP Clonase II Enzyme Mix (Invitrogen, Carlsbad, USA). Gateway compatible pENTR/SD/D/TOPO vectors containing the full length ORFs of ZmTCP01 (clone UT5707), ZmTCP02 (clone UT5978), ZmTCP05 (clone UT1680), ZmTCP12 (clone UT6182), ZmTCP13 (clone UT3439) and ZmTCP18 (clone UT4097) were ordered from The Arabidopsis Information Resource (TAIR) (S4 Table). A codon-optimized version of SAP11MBSP without the sequence corresponding to the signal peptide and DNA sequences corresponding to the TCP domains of ZmTCP9, AtTCP12, AtTCP18 and the AtTCP2 and SAP11 chimeras were gene synthesized by Genscript (New Jersey, USA) with Gateway compatible attL1 and attL2 attachment sites (S4 and S8 Tables) and provided in pMS (Genscript).
Transient expression assays in Arabidopsis thaliana and maize (Zea mays L.) protoplasts
All genes were transferred from the Gateway compatible entry clones into the respective expression vectors with the Gateway LR Clonase II enzyme mix (Invitrogen). Full-length ORFs of all TCPs were cloned into pUGW15 [66] to produce N‐terminally HA‐tagged proteins. The codon-optimized versions of SAP11AYWB and SAP11MBSP without signal peptide sequences were cloned into pUBN-GFP-DEST [67] to produce N‐terminally GFP‐tagged SAP11AYWB and SAP11MBSP. To generate a plasmid for expression of GFP alone, the ccdB cassette of pUBN-GFP-DEST was replaced with a GFP sequence that carries two translational stop codons instead of the translational start codon. The GFP-sequence was amplified from pUBN-GFP-DEST with the gene-specific primers STOP-GFP forward and reverse (S7 Table), cloned into pDONR207 with the Gateway BP Clonase II Enzyme Mix (Invitrogen) and transferred to pUBN-GFP-DEST using the Gateway LR Clonase II Enzyme Mix (Invitrogen).
Isolation and transformation of Arabidopsis and maize protoplasts were performed as described by [68]. Protoplasts were generated from 6-week-old Arabidopsis and four-leaf stage maize plants grown in controlled environmental conditions with a 14h, 22 C°/ 10h, 20°C light / dark period. The maize plants were transferred into dark for five days before protoplast isolation. 600-μl-protoplast-suspensions were transformed with the indicated constructs and placed in the dark for 12h for gene expression. Protoplasts were harvested by mild centrifugation (1 min, 200 x g) and mixed with 20μl 2X sodium dodecyl sulfate (SDS)- polyacrylamide gel electrophorese (PAGE) sample buffer (50 mM Tris/HCl, 10% (w:v) SDS, 50% (v:v) glycerol, 0.02% bromophenolblue, 10% ß-mercaptoethanol, pH = 6.8). Samples were separated in an SDS-PAGE using 15% SDS-polyacrylamide gels and blotted on 0.45μm BA85 Whatman Protran nitrocellulose membranes (Sigma-Aldrich) with the BioRad (Life Science, Hemel Hempstead, UK) minigel and blotting system. Proteins were detected via western blot hybridization with specific antibodies. For detection of GFP-fusion proteins, anti-GFP polyclonal primary antibody (Santa Cruz Biotechnology, Dalla, USA, catalog number: sc-8334, diluted 1:1000) and anti-rabbit-HRP secondary antibody (Sigma-Aldrich, diluted 1:10000) were used. After the anti GFP-antibodies were removed by treatment of the membrane with 0.2 M glycine, 0.1% SDS, 100 mM ß-mercaptoethanol, pH = 2, the HA-fusion proteins were detected on the same blot with anti-HA11 monoclonal primary antibody (Covance, New Jersey, USA, order number: MMS-101P, diluted 1:1000) and anti-mouse-HRP secondary antibody (Sigma-Aldrich, diluted 1:10000).
Yeast Two-Hybrid analyses
All genes were transferred from the above generated Gateway compatible entry clones into the respective Yeast Two-Hybrid vectors with the Gateway LR Clonase II enzyme mix (Invitrogen). The codon-optimized sequences corresponding to mature proteins (without signal peptides) of SAP11AYWB, SAP11MBSP and SAP11 chimeras were transferred into pDEST-GAD-T7 [69]. The TCP sequences encoding for full length TCPs or TCP domains were transferred into the pDEST-GBK-T7 [69]. Saccharomyces cerevisiae strain AH109 (Matchmaker III; Clonetech Laboratories, Mountain View, CA, USA) was transformed using a 96-well transformation protocol [70] and interaction studies were carried out on media depleted of leucine, tryptophan and histidine with addition of 20 mM 3-Amino-1,2,4-triazole (3AT) to suppress auto activation.
Generation and analysis of transgenic A. thaliana lines
The generation and analysis of the 35S::SAP11AYWB Arabidopsis Col-0 lines, was described previously [8]. Idan Efroni (Weizmann Institute of Science, Rehovot, Israel) provided seeds of the 35S::miR319a x 35S::miR3TCP Arabidopsis Col-0 lines described in Efroni et al. [31] and Pilar Cubas (Centro Nacional de Biotecnologia, Madrid, Spain) provided seeds of the brc1 brc2 Arabidopsis Col-0 line described in Aguilar-Martinez et al. [35]. For generation of the 35S::SAP11MBSP Arabidopsis Col-0 lines the codon optimized version of the SAP11MBSP sequence without the sequence corresponding to the signal peptide was transferred from the Gateway compatible entry clone (described above) into the pB7WG2 binary vector using the Gateway LR Clonase II Enzyme Mix (Invitrogen) and Arabidopsis Col-0 plants were transformed using the floral dipping method [71].
Quantitative real time-PCR experiments
SAP11 transcript levels in 35S::SAP11AYWB and 35S::SAP11MBSP A. thaliana plants were quantified in mature leaves of three independent, 5-week-old plants. Total RNAs were extracted from 100 mg snap frozen A. thaliana leaves with TRI-reagent (Sigma Aldrich) and cDNA synthesis was performed from 0.5 μg total RNA using the M-MLV-reverse transcriptase (Invitrogen). cDNA was subjected to qRT-PCR using SYBR Green JumpStart Taq ReadyMix (Sigma-Aldrich) in a CFX96 Touch Real-Time PCR Detection System (Biorad) using gene-specific primers for the SAP11-homologs and Actin 2 (AT3G18780) (S9 Table).
Root length measurements
A. thaliana seeds were sterilized in 5% sodium hypochlorite for 8 minutes and washed five times with sterile water. Seeds were germinated on ½ x MS medium with 0.8% (w/v) agar. Three days after germination, seedlings were transferred to ½ x Hoagland medium [72] with 0.25 mM KH2PO4 containing 1% (w/v) sucrose and 1% (w/v) agar [43]. Plates were placed vertical to allow root growth on the agar surface. After an additional growth period of 10 days seedlings were removed from the plates individually and their root length measured using a ruler.
Generation and analysis of transgenic maize lines
Codon optimized versions of the SAP11AYWB and the SAP11MBSP sequences without sequences corresponding to the signal peptide including a sequence encoding an N-terminal 3xFLAG-tag were synthesized with flanking BamH1 and EcoRI restriction sites (S10 Table) that were used for cloning into the multiple cloning site of the p1u Vector (DNA Cloning Service, Hamburg, Germany). The resulting Ubi::FLAG-SAP11-nos cassette was transferred from p1U into the binary Vector p7i (DNA Cloning Service, Hamburg, Germany) via SfiI restriction sites. Agrobacterium-mediated transformation of maize HiIIAxHiIIB embryos and BASTA (Bayer CropScience, Monheim, Germany) selection of T0 transgenic HiIIAxHiIIB plants was performed by Crop Genetic Systems (CGS) UG (Hamburg, Germany). This resulted in the three independent, transgenic, heterozygous lines of UBI::FLAG-SAP11AYWB 1–3 and the two independent, transgenic, heterozygous lines of UBI::FLAG-SAP11MBSP 1–2. For seed reproduction T0 transgenic plants were crossed with HiIIA plants because the described defects in sexual organs development (Fig 5) impeded self-pollination. Plants were analyzed for production of proteins from transgenes via western blot hybridizations (explained above) with anti-FLAG M2 monoclonal primary antibody (Sigma-Aldrich, order number: F3165, diluted 1:1000) and anti-mouse-HRP secondary antibody (Sigma-Aldrich, diluted 1:10000) and then used for experiments.
Insect fecundity assays
Plants were grown under controlled environmental conditions with a 14h, 22 C°/ 10h, 20°C light / dark period for Arabidopsis and 16h, 26°C/ 8h, 20°C light/dark period for maize. Seven-week-old Arabidopsis and three-week-old maize plants were individually exposed to 10–15 adult M. quadrilineatus or D. maidis insects (7–10 females and 3–5 males) for 3 days. The insects were removed and progeny (nymphs or adults) were counted four weeks later.
RNA-seq analysis
Fully expanded leaves of seven-week-old A. thaliana Col-0 wt and transgenic plants were exposed to five adult M. quadrilineatus (2 males and 3 females) in a single clip cage with one clip-cage per plant. For the generation of non-treated samples, clip-cages were applied without insects. After 48h the areas covered by the clip-cages were harvested, snap frozen in liquid nitrogen and stored at -80°C until further processing for RNA extraction. For maize, complete three-week-old maize HiIIA wild type (WT) or transgenic plants were exposed to 50 adult M. quadrilineatus or D. maidis insects (20 males and 30 females) for 48 hours and the complete above soil plant material was harvested, snap frozen in liquid nitrogen and stored at -80°C until further processing for RNA extraction.
Total RNA was extracted from ground Arabidopsis leaf tissue and from 200 mg ground maize material using the RNeasy plant mini kit with on-column DNase digestion (Qiagen). The RNA-seq data of the A. thaliana experiments were generated at Academia Sinica (Taipei, Taiwan) and at the Earlham Institute (EI, Norwich, UK). The RNA-seq data of all maize experiments were generated at EI. At Academia Sinica, libraries were generated with the llumina Truseq strand-specific mRNA library preparation without size selection, and sequenced on the Illumina HiSeq2500, 125-bp paired-end reads (YOURGENE Bioscience, New Taipei City, Taiwan). Libraries at EI were generated using NEXTflex directional RNA library (HT) preparation (Perkin Elmer, Austin, Texas, USA) and sequencing was done on the Illumina HiSeq4000, 75-bp paired-end reads (EI). To assess if the RNA-seq data for the A. thaliana experiments received from EI and Academia Sinica are comparable, four samples were sequenced at both facilities. Principal Component Analysis (PCA) showed that the samples generated by these two facilities cluster together demonstrating that batch effects are negligible (S12 Fig).
The adapter sequences of the raw RNAseq reads were removed using Trim Galore, version 0.4.4 (https://www.bioinformatics.babraham.ac.uk/projects/trim_galore/). The paired-end reads were aligned to the reference genome (A. thaliana/TAIR 10.23 and Z. mays/AGPv4) with the software TopHat, version 2.1.1 [73]. The number of aligned reads per gene was calculated using HTSeq, version 0.6.1 [74], and data were initially analysed via PCA, using the R/Bioconductor package DESeq2 [75]. Obvious outliers were excluded from the analysis; this amounted to one sample per experiment, as follows: one wild type (WT) Col-0 + M. quadrilineatus sample from the A. thaliana experiment; one Ubi::FLAG-SAP11AYWB + M. quadrilineatus sample from one of the maize experiments; one Ubi::FLAG-SAP11AYWB + D. maidis sample from the other maize experiment; and one Ubi::FLAG-SAP11MBSP sample in common with both experiments (S13 Fig, S1, S5 and S6 Tables). Differential expression analysis was conducted with DESeq2, using the function -contrast- to make specific comparisons. For further analyses we selected genes that satisfy 3 criteria: p value <0.05 after accounting for a 5% false discovery rate (FDR) (Benjamini-Hochberg corrected), mean gene expression value >10 and fold change in expression >2. Cluster analysis was performed on z-score normalized data using the hierarchical method [76].
Transcriptome assemblies of M. quadrilineatus and D. maidis RNA-seq data
RNA-seq data of M. quadrilineatus and D. maidis males and females (~25 million reads each) were downloaded from NCBI, accession number SRP093182 and SRP093180 respectively. The reads were used for de novo assemblies of male and female transcriptomes separately. Reads were trimmed to remove adaptor sequence and low-quality reads using Trim Galore (https://www.bioinformatics.babraham.ac.uk/projects/trim_galore/). Reads over 20-bp in length were retained for downstream analysis. Trimmed reads were de novo assembled using Trinity r20140717 [77] allowing a minimum contig length of 200 bp and minimum k-mer coverage of 2 with default parameters. Assembled contigs were made non-redundant and lowly expressed contigs were filtered with FPKM cut-off 1 using build-in Perl script provided by Trinity. This resulted in 48474 transcripts for male M. quadrilineatus, 44409 transcripts for female M. quadrilineatus, 42815 transcripts for male D. maidis and 59131 transcripts for female D. maidis. These assemblies were used to validate the origin of RNA-seq data by assessing if reads aligning to leafhopper transcripts were present in RNA-seq data derived from plants exposed to the leafhoppers as opposed to those of plants that were not exposed to the leafhoppers.
Supporting information
S1 Fig [tif]
Quantification of the effect of SAP11 on the TCP protein abundance in Arabidopsis protoplasts.
S2 Fig [tif]
Original Western blots of Arabidopsis and maize protoplast destabilization assays presented in and .
S3 Fig [a]
Confocal microscopy images showing SAP11 localizations to cell nuclei of protoplasts.
S5 Fig [a]
Phenotyping of transgenic and Arabidopsis plants.
S6 Fig [a]
CIN-TCP destabilization affects root lengths.
S7 Fig [sub]
Classification of (Sb) TCPs.
S8 Fig [sub]
Classification of (Oz) TCPs.
S9 Fig [zm]
Identification of potential miR319a target sites.
S10 Fig [tif]
Quantification of the effect of SAP11 on the TCP protein abundance in maize protoplasts.
S11 Fig [sub]
Alignment of the TCP domains, consisting of the basic helix-loop-helix regions of (At) TCPs.
S12 Fig [tif]
Principal component analysis (PCA) showing that samples sequenced at different facilities cluster together (batch effect is negligible).
S1 Table [xlsx]
Alignment to SAP11 transgene, M. quadrilineatus transcriptome and A. thaliana genome of RNA-seq data shown in .
S2 Table [xlsx]
List of differentially expressed genes and expression values in RNA-seq experiments of 6 treatments.
S3 Table [xlsx]
List of differentially expressed genes with potential biological functions.
S4 Table [xlsx]
Sequence IDs of TCPs from Zea mays (Zm), Arabidopsis thaliana (At), Sorghum bicolor (Sb) and Oryza sativa (Os).
S5 Table [xlsx]
Alignment to SAP11 transgene, transcriptome and genome of RNA-seq data of -exposed (+Mq) shown in .
S6 Table [xlsx]
Alignment to SAP11 transgene, transcriptome and genome of RNA-seq data of -exposed (+Dm) shown in .
S7 Table [docx]
Oligonucleotide sequences (5´ > 3´) for cloning.
S8 Table [docx]
Synthesized CDS (underlined) flanked by Gateway compatible attL1 and attL2 sites.
S9 Table [docx]
Oligonucleotide sequences (5´ > 3´) for qRT-PCR.
S10 Table [grey]
Nucleotide sequences for gene syntheses of and for expression in Zea mays.
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Štítky
Hygiena a epidemiologie Infekční lékařství LaboratořČlánek vyšel v časopise
PLOS Pathogens
2019 Číslo 9
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