GENETICALLY MODIFIED PLANT AND USES THEREOF

Abstract

The invention provides a genetically modified plant or plant cell transformed with an isolated nucleic acid, wherein the isolated nucleic acid encodes a SYAC1 protein or a protein related to the SYAC1 protein or a protein with at least about 80%, 85%, 90%, 95%, 98%, 99% or more sequence identity protein with a SYAC1 protein. The invention also provides a genetically modified plant or plant cell engineered to express a reduced level, or no, SYAC1 protein. A genetically modified plant according to the invention may be more resistant to infection by Plasmodiophora brassicae. Alternatively, the invention provides a genetically modified plant with increased levels of SYAC1 protein or related protein which is more receptive to beneficial microorganisms and/or grows more effectively on land contaminated with heavy metals.

Claims

1. A genetically modified plant or plant cell transformed with an isolated nucleic acid, wherein the isolated nucleic acid encodes a SYAC1 protein or a protein related to the SYAC1 protein or a protein with at least about 80%, 85%, 90%, 95%, 98%, 99% or more sequence identity with a SYAC1 protein.

2. A genetically modified plant or plant cell transformed with an isolated nucleic acid, wherein the isolated nucleic acid comprises or consists of sequence selected from the group consisting of Seq ID no: 1, Seq ID no: 2, Seq ID no: 3, Seq ID no: 4, Seq ID no: 5, Seq ID no: 6, Seq ID no: 7 or Seq ID no: 8, or a sequence that has at least 80%, 85%, 90%, 95%, 98%, 99% or more sequence identity with one of Seq ID no: 1, Seq ID no: 2, Seq ID no: 3, Seq ID no: 4, Seq ID no: 5, Seq ID no: 6, Seq ID no: 7 or Seq ID no: 8.

3. The genetically modified plant or plant cell of claim 1 wherein the isolated nucleic acid encodes a polypeptide having the sequence of Seq ID no: 9, Seq ID no: 10, Seq ID no: 11, Seq ID no: 12, Seq ID no: 13, Seq ID no: 14, Seq ID no: 15 or Seq ID no: 16, or a sequence that has at least about 80%, 85%, 90%, 95%, 98%, 99% or more sequence identity with one of Seq ID no: 9, Seq ID no: 10, Seq ID no: 11, Seq ID no: 12, Seq ID no: 13, Seq ID no: 14, Seq ID no: 15 or Seq ID no: 16.

4. The genetically modified plant or plant cell of claim 1 wherein the expression of the isolated nucleic acid is under the control of the SYAC1 promoter of Seq ID no: 17, or a sequence that has at least about 80%, 85%, 90%, 95%, 98%, 99% or more sequence identity with Seq ID no: 17, or a functional fragment thereof.

5. The genetically modified plant or plant cell of claim 1 which expresses an elevated level of SYAC1 protein or a protein related to the SYAC1 protein, compared to a wild type plant or plant cell.

6. (canceled)

7. The genetically modified plant or plant cell of claim 1 which has one or more of the characteristics selected from the group consisting of a) an increased level of hemicellulose in the cell walls compared to a wild type plant of plant cell; b) less rigid cell walls compared to a wild type plant of plant cell; c) an increased tolerance to growth on contaminated soil compared to a wild type plant or plant cell; and d) altered susceptibility to one or more microbes.

8. The genetically modified plant or plant cell of claim 1 wherein the plant or plant cell is more resistant to disease caused by Plasmodiophora brassicae than a wild type plant or plant cell.

9. The genetically modified plant or plant cell of claim 8 wherein the plant or plant cell has a reduced level of, or no, SYAC1 protein or a protein related to the SYAC1 protein, compared to a wild type plant or cell.

10. The genetically modified plant or plant cell of claim 1 wherein the plant or plant cell has an increased susceptibility to a symbiont or to other beneficial microorganisms.

11. The genetically modified plant or plant cell of claim 10 wherein the plant or plant cell has an elevated level of SYAC1 protein or a protein related to the SYAC1 protein, compared to a wild type plant.

12. The genetically modified plant or plant cell of claim 1 wherein the plant is a monocotyledonous plant or a dicotyledonous plant.

13. The genetically modified plant or plant cell of claim 1 where the spatial and/or temporal expression of the SYAC1 protein or a protein related to the SYAC1 protein, is controlled by a hormone, such as auxin and/or a cytokinin, and/or the copper concentration.

14. (canceled)

15. (canceled)

16. A seed produced by a genetically modified plant or plant cell of claim 1.

17.-20. (canceled)

21. A promoter comprising or consisting of the sequence of SEQ ID NO: 17, or a sequence that has at least about 80%, 85%, 90%, 95%, 98%, 99% or more sequence identity with SEQ ID NO: 17, or a functional fragment thereof.

22.-28. (canceled)

Description

[0063] Preferred embodiments of the present invention will now be described, merely by way of example, with reference to the following figures and examples.

[0064] FIG. 1—is a simplified flow chart which shows the process of the production of bioethanol from plant material. Source: US DOE. June 2007. Biofuels: Bringing Biological Solutions to Energy Challenges, US Department of Energy Office of Science, reproduced with the permission of the US Department of Energy Genomic Science Program—https://genomicscience.energy.gov.

[0065] FIG. 2—shows the developmentally specific expression of SYAC1 and its regulation upon hormonal treatment. (A-C) Expression of SYAC1 in 5-day-old roots is synergistically upregulated after 6 hours treatment with 1 μM auxin and 10 μM cytokinin. SYAC1 expression in Arabidopsis roots monitored by RT-qPCR (A). Error bars represent standard error. Significant differences are indicated as ***P<0.001 (t test). Expression of pSYAC1:GUS (B) and pSYAC1:nlsGFP (C) upon hormonal treatment. (D-H) Expression pattern of SYAC1 from mature embryo till 4-day-old seedling. Mature embryo (D), 2, 3 and 4-day-old seedling (E-G); dark grown hypocotyl and apical hook of 3-day-old seedling (H). Scale bar 50 μm (B, E-G), 20 μm (C), 200 μm (D) and 100 μm (H). (A-C) Expression of SYAC1 in 5-day-old roots is synergistically upregulated after 6 hours treatment with 1 μM auxin and 10 μM cytokinin.

[0066] SYAC1 expression in Arabidopsis roots monitored by RT-qPCR (A). Error bars represent standard error. Significant differences are indicated as ***P<0.001 (t test). Expression of pSYAC1:GUS (B) and pSYAC1:nlsGFP (C) upon hormonal treatment. (D-H) Expression pattern of SYAC1 from mature embryo till 4-day-old seedling. Mature embryo (D), 2, 3 and 4-day-old seedling (E-G); dark grown hypocotyl and apical hook of 3-day-old seedling (H). Scale bar 50 μm (B, E-G), 20 μm (C), 200 μm (D) and 100 μm (H).

[0067] FIG. 3—shows the results of Fourier transform infrared spectroscopy (FT-IR) of 4 day old etiolated hypocotyls illustrating the differences in cell wall composition in control plant cells: Arabidopsis thaliana (L.) Heynh ecopyte Columbia and in SYAC1-HAox plant cells (plant cells transformed with the SYAC1 gene).

[0068] FIG. 4—shows the results of apparent Young modules measured by Atomic Force Microscopy in 4-day-old etiolated hypocotyls illustrating the differences in cell wall physical properties in control plant cells: Arabidopsis thaliana (L.) Heynh ecopyte Columbia and in SYAC1-GFPox plant cells (plant cells transformed with the SYAC1 gene). Significant differences are indicated as *P<0.05 (t-test).

[0069] FIGS. 5A and 5B—shows the increase in hemicellulose concentration in the cell wall of plant cells expressing SYAC1 as determined by antibody staining. In particular, FIG. 5A shows the immunolocalisation of the hemicellulose xyloglucan using the LM 15 antibody and shows the change, and in particular, the increase, in xyloglucan localisation in the cell wall of cells of the SYAC1-GFPox line. FIG. 5B shows the quantification of xyloglucan immunodetected by LM 15 (xyloglucan antibody) in root meristem. Quantification was performed by measurement of membrane signal in cortex and epidermal cells, respectively. Signal in approximately 10 cells in a minimum of 10 roots was measured using ImageJ software. Statistical significance was evaluated by Student's t-test.

[0070] FIG. 5C shows that the amount of galacturonic acid (backbone of pectin structure) extracted from 4-day-old etiolated hypocotyls in SYAC1ox lines is significantly lower. (n=6, average±SE). Wild-type I represents corresponding control to SYAC-GFPox and GFP-SYAC1ox. Wild-type II was isolated from syac1-5 heterozygote population.

[0071] FIG. 6—shows the effect of SYAC1 expression on the α-amylase secretion index. The error bars indicate standard error calculated from 4 independent measurements. Significant differences are indicated as **P<0.01, and ***P<0.001 (t-test).

[0072] FIG. 7—shows the effect of SYAC1 expression on mucilage secretion. In particular, the figure uses ruthenium red to show that SYAC1 expression inhibits mucilage secretion. Scale bar 200 μm.

[0073] FIG. 8—shows that SYAC1 expression renders root plant more tolerant to elevated copper concentrations. Plants were grown for 5 days on standard MS media and then transferred to media containing 50 μM CuSO4. CuSO4 is shown to trigger swelling of cells at the root tip of wild type control plants, but not SYAC1-GFPox plants. These results demonstrate that SYAC1 expression renders plant cells more tolerant to elevated copper.

[0074] FIG. 9—shows that SYAC1 expression reduces the uptake of copper and iron. In particular, the results demonstrate that SYAC1 expression significantly reduces the amount of Cu or Fe taken up by plant roots. Wild type Col-0 and the SYAC1 overexpressor lines (HA-SYAC1ox) were grown for 1 week in standard MS media on agar plates. Fresh roots were collected and rinsed in deionized water during the sampling and stored in Falcon tubes. To remove the copper in the cell apoplast, fresh roots samples were immediately washed once in 40 ml 1 mM HCl solution (shaking end over end) for 3 mins. First washing solution was removed and samples were additionally washed in 0.01M HCl for 5 mins. Finally, root samples were rinsed in 40 ml deionized H.sub.2O (Chaignon and Hinsinger, J. Environ. Qual., 2003). Samples were dried overnight in Falcon tubes at 65° C. in oven. For analysis of metal content, sample digestion has been performed at 150° C. for 4 h in a mixture of 65% HNO3 and 30% H2O2 in a ratio of 5:1 (v/v). Copper and iron were measured by detecting two isotopes, Cu-63 and Cu-65 or Fe-54 and Fe-57 respectively in a Perkin Elmer Elan DRCe 9000 (Rosenkranz et al., Waste Manag., 2018).

[0075] FIG. 10A—shows that SYAC1 expression is interfering with pectin secretion in tobacco pollen tubes (Nicotiana tabacum; ecotype Samsun N).

[0076] FIG. 10B shows the quantification of pectin detected by Ruthenium red staining in tobacco pollen tubes Nicotiana tabacum; ecotype Samsun N. Quantification was performed by measurement of ruthenium red intensity in the growing tips of tobacco pollen tubes. Signal in n=48 control pollen tubes and n=66 pollen tubes expressing SYAC1 was measured using ImageJ software. Statistical significance was evaluated by Student's t-test.

[0077] FIG. 11 shows the impact of SYAC1 on plant sensitivity to Plasmodiophora brassicae infection. Sensitivity of syac1-3 and syac1-5 mutant alleles, and SYAC1ox lines to pathogen infection. 5-scale classification was used to evaluate disease severity: 0 (no symptoms), 1 (very small galls mainly on lateral roots and that do not impair the main root), 2 (small galls covering the main root and few lateral roots), 3 (medium to large galls, also including the main root; plant growth might be impaired), and 4 (severe galls on lateral root, main root, or rosette; fine roots completely destroyed; plant growth is reduced). Inoculation was performed with 10.sup.4, 10.sup.5 and 10.sup.6 spores per mL. Significant differences between datasets are indicated by different letters.

[0078] FIG. 12 also shows the impact of SYAC1 on plant sensitivity to Plasmodiophora brassicae infection. Shoots of wild-type, syac1-3, syac1-5 and SYAC1ox 28 days after P. brassicae inoculation are depicted. Inoculation was performed with 10.sup.4, 10.sup.5 and 10.sup.6 spore per mL.

[0079] FIGS. 13A, 13B and 13C show the effect of the expression of SYAC1 paralogues on α-amylase secretion (the effect of SYAC1 is shown in FIG. 6). α-Amylase secretion assay was performed in Arabidopsis mesophyll protoplasts. SYAC1 and its paralogues were transiently co-expressed with α-Amylase (Amy) and α-Amylase derivatives fused to C-terminal vacuolar sorting (Amy-spo) and Endoplasmatic reticulum (ER) retention (Amy-HDEL) motif. FIG. 13A shows the effect of expression of SYAC1 and its paralogues on the α-amylase (Amy) secretion index. FIG. 13B shows the effect of expression of SYAC1 and its paralogues on the α-amylase fused to C-terminal vacuolar sorting motif (Amy-spo) secretion index. FIG. 13C shows the effect of expression of SYAC1 and its paralogues on the α-amylase fused to Endoplasmatic reticulum sorting motif (Amy-HDEL) secretion index. Secretion index was measured as a ratio of the α-Amylase activity in the medium and in the cells. The error bars indicate standard error calculated from 4 independent measurements. Significant differences are indicated as *P<0.05 (t-test).

PLANT MATERIAL AND GROWTH CONDITIONS

[0080] Seeds of Arabidopsis were plated and grown on square plates with solid half strength Murashige and Skoog (MS) medium (Duchefa) supplemented with 0.5 g L.sup.−1 MES, 10 g L.sup.−1 Suc, 1% agar and pH adjusted to 5.9. The plates were incubated at 4° C. for 48 h to synchronize seed germination and then vertically grown under a 16:8 h day/night cycle photoperiod at 21° C.

[0081] The syac1-1 (salk_151420C, Col-0, KAN.sup.R) and syac1-2 (salk_151662B, Col-0, KAN.sup.R) T-DNA insertion lines were obtained from the Salk Institute. The syac1-3 (GABI-KAT 760F05, Col-0, SUL.sup.R) and syac1-4 (GABI-KAT 961C03, Col-0, SUL.sup.R) T-DNA insertion lines were obtained from the GABI KAT seed collection. The syac1-5 CRISPR line was prepared in collaboration with the VBCF Protein Technologies Facility (www.vbcf.ac.at) (see below). The genetically modified fluorescent-protein marker lines in Col-0 background have been described elsewhere: mCherry tagged wave line 6, 9, 13, 18, 25, 29, 34, 127, 129, 131, 138 (Geldner et al., 2009, Plant J. 59, 169-178), SYP61:SYP61-CFP (Drakakaki et al., 2012, Cell Res. 22, 413-424). The echidna mutant has been described in (Gendre et al., 2011, PNAS 108, 8048-8053) and yip4a-2 yip4b-1 in (Gendre et al., 2013, The Plant Cell 25, 2633-2646). Seeds of Arabidopsis were plated and grown on square plates with solid half strength Murashige and Skoog (MS) medium (Duchefa) supplemented with 0.5 g L.sup.−1 MES, 10 g L.sup.−1 Sucrose, 1% agar and pH adjusted to 5.9. The plates were incubated at 4° C. for 48 h to synchronize seed germination and then vertically grown under a 16:8 h day/night cycle photoperiod at 21° C. Cytokinin and auxin treatments were performed with the N6-benzyladenine cytokinin derivative (Sigma) and Naphthaleneacetic acid (Sigma), respectively. Short treatments (6 hours) for GUS/GFP expression were performed with 10 μM cytokinin and 1 μM auxin (unless indicated differently). For root growth transient assay 0.1 μM cytokinin and 0.05 μM auxin was used. Estradiol treatment was performed with β-Estradiol (Sigma).

[0082] Cloning and Generation of Genetically Modified Lines

[0083] All cloning procedure was conducted by using Gateway™ (Invitrogen) technology; with the sequences of all used vectors available online (https://gateway.psb.ugent.be/). To generate lines with constitutive overexpression of SYAC1 (SYAC1-GFPox, SYAC1-HAox, HA-SYAC1ox), SYAC1 ORF sequence was amplified and fused through a linker (4 Glycines and 1 Alanine) to GFP or HA tag. The fragments were first introduced into pDONR221 and then into pB2GW7,0 vector. All genetically modified plants were generated by the floral dip method (Clough and Bent, 1998, J. Cell Mol. Biol. 16, 735-743), and transformants were selected on plates with appropriate antibiotic.

[0084] For promoter analysis of SYAC1, an upstream sequence of 2522 bp was amplified by PCR and introduced into the pDONRP4-P1R entry vector. Then transcriptional lines (pSYAC1:GUS, pSYAC1:nlsGFP) were created: for pSYAC1:GUS, an LR reaction with SYAC1 promoter in pDONORP4-P1R, pEN-L1-S-L2,0 and pK7m24GW,0 vectors was performed. For pSYAC1:nlsGFP line, an LR reaction with SYAC1 promoter in pDONORP4-P1R, pEN-L1-NF-L2,0 and pB7m24GW,0 was performed. To generate overexpressor and inducible lines (SYAC1-GFPox, SYAC1-HAox, HA-SYAC1ox, pEST:SYAC1-GFP, pEST:SYAC1), SYAC1 ORF sequence with or without STOP codon was amplified and fused through a linker (4 Glycines and 1 Alanine) to GFP or HA tag. The fragments were first introduced into pDONR221 and then into pB2GW7,0 (overexpressor lines), p2GW7,0 (protoplast expression assays), pMDC7 (estradiol inducible line). For GFP-SYAC1ox genetically modified line SYAC1 ORF was amplified, introduced to pDONR221 and to the pB7FWG2.0 destination vector. To generate translational fusion line pSYAC1:gSYAC1-GFP, SYAC1 promoter was amplified together with the genomic fragment of the SYAC1 gene, cloned into pDONRP4-P1R and together with pEN-L1-F-L2,0 introduced into pB7m24GW,3. All genetically modified plants were generated by the floral dip method (Clough and Bent, 1998) in Columbia (Col-0) background and transformants were selected on plates with appropriate antibiotic.

[0085] Generation of CRISPR/Cas9 Line

[0086] Design of the gRNA for SYAC1 gene, molecular cloning and plant transformation was done in collaboration with VBCF Protein Technologies Facility (www.vbcf.ac.at). Design, specificity and activity of gRNA: GATGGTCAGCAACCACACGA (Seq ID no: 18) was performed using online available tools: http://cbi.hzau.edu.cn/cgi-bin/CRISPR and http://www.broadinstitute.org/rnai/public/analysis-tools/sgrna-design. gRNA was cloned into pGGZ003 CRISPR/Cas9 destination vector. Transformants resistant to an appropriate antibiotic were selected, genomic sequence of SYAC1 amplified and sequenced. Individual mutant lines with a single base pair insertion in the coding sequence (90 bps after the ATG—at the place of gRNA binding) were selected. Plants were propagated to obtain homozygote lines and CRISPR/Cas9 cassette was outcrossed.

[0087] Quantitative RT-PCR

[0088] RNA was extracted (RNeasy kit (Qiagen)) from roots of 6-day-old plants under all conditions (untreated, 1 μM auxin, 10 μM cytokinin and both together for 6 h). A DNase treatment with the RNase-free DNase Set (Qiagen) was carried out for 15 min at 25° C. Poly(dT) cDNA was prepared from 1 μg of total RNA with the iScript cDNA Synthesis Kit (Biorad) and analyzed on a LightCycler 480 (Roche Diagnostics) with the SYBR Green I Master kit (Roche Diagnostics) according to the manufacturer's instructions. SYAC1 expression was quantified with specific primer pair Fw: ACTTCTGGTTATGTTTGGCTCTCC (Seq ID no: 19) and Rv: ACACATATGACCACAGGCGTAAG (Seq ID no: 20). All PCRs were performed in 3 technical replicates and the experiments were repeated three times with similar results. Expression levels were first normalized to CDKA1 (Fw: ATTGCGTATTGCCACTCTCATAGG (Seq ID no: 21) and Rv: TCCTGACAGGGATACCGAATGC (Seq ID no: 22)) expression levels and then to the respective expression levels in untreated or in wild-type plants.

[0089] Phenotypic Analysis

[0090] For root and hypocotyl length analysis, seedlings were photographed and lengths were measured with ImageJ software (https://imagej.nih.gov/ij/). About 20-30 seedlings were processed and 3 independent experiments were performed.

[0091] Histochemical and Histological Analysis

[0092] To detect β-Glucuronidase (GUS) activity, mature embryos and 2 to 4-day-old seedlings were incubated in reaction buffer containing 0.1M sodium phosphate buffer (pH 7), 1 mM ferricyanide, 1 mM ferrocyanide, 0.1% Triton X-100 and 1 mg m1.sup.−1 X-Gluc for 12 h in dark at 37° C. Afterwards, chlorophyll was removed by destaining in 70% ethanol and seedlings were cleared as described (Malamy and Benfey, 1997, Development 124, 33-44). In brief, seedlings were incubated in a solution containing 4% HCl and 20% methanol for 10 min at 65° C., followed by 10 min incubation in 7% NaOH/60% ethanol at room temperature. Next, seedlings were rehydrated by successive incubations in 60, 40, 20 and 10% ethanol for 15 min, followed by incubation (15 min up to 2 h) in a solution containing 25% glycerol and 5% ethanol. Finally, seedlings were mounted in 50% glycerol. GUS expression was monitored by differential interference contrast microscopy (Olympus BX53).

[0093] Immuno labeling in roots (4 to 5-day-old seedlings) was performed using an automated system (Intavis in situ pro) according to published protocol (Sauer et al., 2006, Nature Protocols. Nat. Protocols 1, 98-103). Roots were fixed in 4% paraformaldehyde for 1 h in vacuum at room temperature. Afterwards, seedlings were incubated for 30-45 min in PBS (2.7 mM KCl, 137 mM NaCl, 4.3 mM Na.sub.2HPO.sub.42H.sub.2O and 1.47 mM KH.sub.2PO.sub.4, pH 7.4) containing 2% driselase (Sigma), and then in PBS supplemented with 3% NP40 and 20% DMSO. After blocking with 3% BSA in PBS, samples were incubated with primary antibody for 2 hours. Antibody dilutions were rabbit anti-BIP2 (1:200) (Agrisera AS09481), rabbit anti-SEC21 (1:800) (Agrisera AS08327), rabbit anti-ARF1 (1:600) (Agrisera AS08325), rabbit anti-SYP61 (1:200) (Sanderfoot et al., 2001), rabbit anti-ECH (1:600) (kindly provided by R. P. Bhalerao, Umea Plant Science Centre), rabbit anti-ARA7+RHA1 1:1 (1:100) (Haas et al., 2007), rabbit anti-PIN1 (1:1000) (Paciorek et al., 2005), rabbit anti-PIN2 (1:1000) (provided by C. Luschnig, University of Natural Resources and Life Sciences, Vienna) and mouse anti-GFP (1:600) (Sigma G6539). Secondary antibody incubation was carried on for 2 h. Anti-mouse-Alexa 488 (Life Technologies, 1252783) and Cy3-conjugated anti-rabbit antibody (Sigma, C2306) were diluted 1:600 in blocking solution. Samples were mounted in solution containing 25 mg mL.sup.−1 DABCO (Sigma) in 90% glycerol, 10% PBS, pH 8.5. Signal was monitored using a confocal laser scanning microscope (LSM 700, Zeiss). Images were analyzed by using ImageJ software.

[0094] Co-Localization Analysis

[0095] Pearson's correlation coefficient (R) was used for co-localization analyses: the analysis is based on the pixel intensity correlation over space and was performed using Image J software. After splitting the two channels, region of interest (ROI) was identified. For this analysis, 1 cell was considered as 1 ROI; in every root approximately 5 cells (5 ROIs) were measured and a minimum of 10 roots were used. Co-localization plug-in using an automatic threshold was used to obtain Rcoloc value, which represent Pearson's correlation coefficient.

[0096] Confocal Imaging and Image Analysis

[0097] Zeiss LSM 700 confocal scanning microscope using either 20× or 40× (water immersion) objectives were employed to monitor expression of fluorescent reporters. GFP (YFP) and Cy3 signals were detected either at 488 nm excitation/507 nm emission or 550 nm excitation/570 nm emission wavelength, respectively. Quantification of immunodetected PIN1 and PIN2 expression in root meristems was performed by measurement of membrane signal in cortex and epidermal cells, respectively. Signal in approximately 10 cells in a minimum of 10 roots was measured using ImageJ software. Statistical significance was evaluated by Student's t-test.

[0098] Transient Expression in Root Suspension Culture Protoplasts

[0099] The transient expression assays were performed on 4-days-old Arabidopsis root suspension culture by PEG mediated transformation. Protoplasts were isolated in enzyme solution (1% cellulose (Serva), 0.2% Macerozyme (Yakult), in B5-0.34M glucose-mannitol solution (2.2 g MS with vitamins, 15.25 g glucose, 15.25 g mannitol, pH to 5.5 with KOH) with slight shaking for 3-4 h, and afterwards centrifuged at 800 g for 5 min. The pellet was washed and resuspended in B5 glucose-mannitol solution to a final concentration of 4×10.sup.6 protoplasts/mL. DNAs were gently mixed together with 50 μL of protoplast suspension and 60 μL of PEG solution (0.1M Ca(NO.sub.3).sub.2, 0.45M Mannitol, 25% PEG 6000) and incubated in the dark for 30 min. Then 140 μL of 0.275M Ca(NO.sub.3).sub.2 solution was added to wash off PEG, wait for sedimentation of protoplasts and remove 240 μLof supernatant. The protoplast pellet was resuspended in 200 μL of B5 glucose-mannitol solution and incubated for 16 h in the dark at room temperature. Transfected protoplasts were mounted on the slides and viewed with Zeiss LSM 700 confocal scanning microscope.

[0100] Transient Expression in Arabidopsis Mesophyll Protoplasts

[0101] Mesophyll protoplasts were isolated from rosette leaves of 4-week-old Arabidopsis plants grown in soil under controlled environmental conditions in a 16:8 h light/dark cycle at 21 C. Protoplasts were isolated and transient expression assays were carried out as described (Wu et al., 2009, Plant Methods 5, 16).

[0102] Transient Expression in Tobacco Pollen Tubes

[0103] SYAC1 was transiently expressed in tobacco (Nicotiana tabacum) pollen tubes under a pollen-specific Lat52 promoter, exactly as previously described (Ischebeck et al., 2008, Plant Cell 20, 3312-3330).

[0104] Co-Immunoprecipitation (Co-IP) Assays.

[0105] For the Co-IP assays, proteins were expressed in root suspension culture protoplasts (see above) and extracted from the cell pellet as described previously (Cruz-Ramirez et al., 2012, Cell 150, 1002-1015); vectors containing ECH-HA and YIP4a-Myc were kindly provided by R. P. Bhalerao, Umea Plant Science Centre. 100 μg total protein extract was incubated in a total volume of 100 μL extraction buffer containing 150 mM NaCl and 1 μg anti-GFP (JL-8, Clontech) or 1.5 μg anti-cMyc (clone 9E10, Covance). After 2 h, 15 μL ProteinG-Magnetic Beads (BIO-RAD), which were previously equilibrated in TBS buffer we added and this mixture was further incubated for another 2 h on a rotating wheel at 4° C. The beads were then washed in 3×500 μL washing buffer (1×TBS, 5% glycerol, 0.1% Igepal CA-630) and eluted by boiling in 25 μL 1.5× Laemmli sample buffer. Proteins were then resolved with SDS-PAGE and blotted to PVDF transfer membrane (Millipore). The presence of the proteins of interest was tested by immunodetection using rat anti-HA-peroxidase (3F10, Roche).

[0106] Bimolecular Fluorescence Complementation (BiFC) Assay

[0107] To generate constructs for BiFC assay, the ORFs for SYAC1, YIP4a, YIP4b, YIP5b, ECH, KCR1 and PHB4 proteins were cloned into the pDONRZeo vector. Next, the ORFs were transferred from their respective entry clones to the gateway vector pSAT4-DEST-n(174)EYFP-C1 (ABRC stock number CD3-1089) or pSAT5-DEST-c(175-end)EYFP-C1 (ABRC stock number CD3-1097), which contained the N-terminal 174 amino acids of enhanced yellow fluorescent protein (EYFP.sup.N) or the C-terminal 64 amino acids of EYFP (EYFP.sup.C), respectively. The fusion constructs encoding cEYFP-SYAC1 and nEYFP-YIP4a, nEYFP-YIP4b, nEYFP-YIP5b, nEYFP-ECH, nEYFP-KCR1 or nEYFP-PHB4 proteins were mixed at a 1:1 ratio and transfection of root suspension culture protoplasts (see above) was performed. SYAC1 in P2YGW7 was used as a positive control.

[0108] Yeast Two-Hybrid Assays.

[0109] Yeast two-hybrid assay was performed using the GAL4-based two-hybrid system (Clontech). Full-length SYAC1 and YIP4a, YIP4b, YIP5b, ECH, KCR1, DSK2, PHB4 ORFs were cloned into pDEST-GADT7 and pDEST-GBKT7 (Clontech) to generate the constructs AD-SYAC1 and BD-YIP4a (YIP4b, YIP5b, ECH, KCR1, DSK2, PHB4). The constructs were transformed into the yeast strain PJ69-4A with the lithium acetate method. The yeast cells were grown on minimal medium (-Leu/-Trp), and transformants were plated (minimal medium, -Leu/-Trp/-His without or with increasing concentration of 3-Amino-1,2,4-trizol) to test the protein interactions.

[0110] α-Amylase Enzymatic Assay.

[0111] α-Amylase assays and calculations of the secretion index were performed as described (Früholz and Pimpl, 2017, In Plant Protein Secretion, (Humana Press, New York, N.Y.), pp. 171-182); α-Amylase expression constructs were kindly provided by P. Pimpl and transfections were performed in Arabidopsis mesophyll protoplasts (see above). α-Amylase activity was measured with a kit Ceralpha (Megazyme). The reaction was performed in a microtiter plate at 37° C. with 30 μL of extract and 30 μL of substrate. The reaction was stopped by the addition of 150 mL of stop buffer. The absorbance was measured at a wavelength of 405 nm. Experiment was performed three times with four replicates.

[0112] Real-Time Analysis and Statistics of the Apical Hook Development

[0113] Development of seedlings was recorded at 1-h intervals for 5 days at 21° C. with an infrared light source (880 nm LED; Velleman, Belgium) by a spectrum-enhanced camera (EOS035 Canon Rebel Xti; 400DH) with built-in clear wideband-multicoated filter and standard accessories (Canon) and operated by EOS utility software. Angles between hypocotyl axis and cotyledons were measured by ImageJ software. At least 10 seedlings with synchronized germination were processed and the experiment was repeated three times with similar results. For more details see (Zhu et al., 2017, In Plant Hormones, J. Kleine-Vehn, and M. Sauer, eds. (New York, N.Y.: Springer New York), pp. 1-8).

[0114] AFM Measurements and Apparent Young's Modulus Calculations

[0115] The AFM data were collected and analyzed as described elsewhere with minor changes (Peaucelle et al., 2015 Curr. Biol. CB 25, 1746-1752). To examine extracellular matrix properties the turgor pressure was suppressed by immersing seedling in a hypertonic solution (0.55 M mannitol). 4 days-old seedlings grown in darkness were placed on petri dishes filled with 1% Agarose and 10% Mannitol and immobilized by low melting agarose (0.7% with 10% Mannitol). The focus on the atomic force microscope was set on the anticlinal (perpendicular to the organ surface) cell walls and its extracellular matrix. To ensure proper indentations (especially in the bottom of the dome shape between two adjacent cells regions), cantilevers with long pyramidal tip (14-16 μm of pyramidal height, AppNano ACST-10), with a spring constant of 7.8 N/m were used. The instrument used was a JPK Nano-Wizard 3.0 and indentations were kept to <10% of cell height. Three scan-maps per sample were taken over an intermediate region of the hypocotyls, using a square area of 25×25 μm, with 16×16 measurements, resulting in 1792 force-indentation experiments per sample. The lateral deflection of the cantilever was monitored and in case of any abnormal increase the entire data set was discarded. The apparent Young's modulus (EA) for each force-indentation experiment was calculated using the approach curve (to avoid any adhesion interference) with the JPK Data Processing software (JPK Instruments AG, Germany). To calculate the average EA for each anticlinal wall, the total length of the extracellular region was measured using masks with Gwyddion 2.45 software (at least 20 points were taken in account). The pixels corresponding to the extracellular matrix were chosen based on the topography map. For topographical reconstructions, the height of each point was determined by the point-of-contact from the force-indentation curve. A total of 12-14 samples were analyzed. A standard t-test was applied to test for differences between genotypes.

[0116] Ruthenium Red Staining

[0117] Mature seeds were incubated in 0.01% (w/v) aqueous solution of Ruthenium red. Seeds were mounted in water and viewed using a DIC Olympus BX53 microscope. Nicotiana tabacum pollen tubes were stained as previously described (Ischebeck et al., 2008, Plant Cell 20, 3312-3330)

[0118] Tandem Affinity Purification (TAP)

[0119] Tandem affinity purification assay was performed in Arabidopsis cell suspension culture as described (Van Leene et al., 2015, Nature Protocols 10, 169-187) with minor modifications. Briefly, SYAC1 was produced as N-terminally tagged GS.sup.TEV fusion in PSB-D cell culture. Extract and binding were performed with 1% digitonin added to the standard buffer. Protein interactors were identified by mass spectrometry using an LTQ Orbitrap Velos mass spectrometer. Proteins with at least two matched high confident peptides were retained. Background proteins were filtered out based on frequency of occurrence of the co-purified proteins in a large dataset containing 543 TAP experiments using 115 different baits.

[0120] Cell Wall Analyses

[0121] Analyses were performed on 4 days old dark grown hypocotyls using an alcohol-insoluble residue (AIR) prepared as follows. Seeds of Arabidopsis were plated and grown on square plates with Milieu Arabidopsis medium (Duchefa) supplemented with 0.32 g L.sup.−1 CaNO.sub.3, 10 g L.sup.−1 Suc, 0.8% agar and pH adjusted to 5.75. The plates were incubated at 4° C. for 48 h to synchronize seed germination and then vertically grown in darkness at 18° C. Freshly collected samples were submerged into 96% ethanol, grinded and incubated for 30 min at 70° C. The pellet was then washed twice with 96% ethanol and once with acetone. The remaining pellet of alcohol insoluble residues (AIR) was dried in a fume hood overnight at room temperature. Dry weight of each sample was measured. After saponification of the AIR (1-4 mg) with 0.05 M NaOH, supernatant containing methyl ester released from the cell wall was separated from the pellet with polysaccharides. Pectins were extracted from the pellet with 1% ammonium oxalate at 80° C. for two hours as described (Krupková et al., 2007, Plant J. 50, 735-750; Mouille et al., 2007, Plant J. 50, 605-614; Neumetzler et al., 2012, PLoS ONE 7, e42914). Galacturonid acid was then quantified by colorimetry using meta-hydroxydiphenyl-sulfuric acid method as described (Blumenkrantz and Asboe-Hansen, 1973, Analytical Biochemistry 54, 484-489). Methyl ester was quantified from NaOH supernatant with a colorimetric assay using enzymatic oxidation of methanol (Klavons and Bennett, 1986, J. Agric. Food Chem. 34, 597-599). The monosaccharide composition of the non-cellulosic fraction was determined by hydrolysis of 100 μg AIR with 2 M TFA for 1 h at 120° C. After cooling and centrifugation, the supernatant was dried under a vacuum, resuspended in 200 μL of water and retained for analysis. All samples were filtered using a 20-μm filter caps, and quantified by HPAEC-PAD on a Dionex ICS-5000 instrument (ThermoFisher Scientific) as described (Fang et al., 2016, The Plant Cell 28, 2991-3004).

[0122] Fourier Transform Infrared Spectroscopy (FT-IR)

[0123] Spectra were recorded from the 4 days old dark grown hypocotyls sections in transmission mode on a Bruker Tensor 27 spectrometer equipped with a Hyperion 3000 microscopy accessory and a liquid N.sub.2 cooled 64×64 mercury cadmium telluride (MCT) focal plane array (FPA) detector. The entire setup was placed on a vibration-proof table. Spectra were recorded in the region 900-3900 cm.sup.−1, with 4 cm.sup.−1 spectral resolution and 32 scans co-added in double sided, forward-backward mode. FPA frame rate was 3773 Hz and integration time 0.104 ms, with offset and gain optimized for each sample between 180-230 and 0-1, respectively. A low pass filter and an aperture of 6 mm were used. 4 hypocotyls for each line were used and 5 spectra from each of 3 different regions were used in the analyses. Background was recorded on a clean, empty spot on the CaF.sub.2 carrier (Crystran Ltd, UK) and automatically subtracted. Fourier transformation was carried out using a zero filling factor of two, and Blackman-Harris 3-term apodization function. Phase correction was set to the built-in Power mode with no peak search and a phase resolution of 32. White light images were recorded with a Sony ExwaveHAD color digital video camera mounted on the top of the microscope and exported as jpg files. Spectra were recorded using OPUS (version 6.5 and 7, Bruker Optics GmbH, Ettlingen, Germany), cut to the fingerprint region of 900-1800 cm.sup.−1 and exported as .mat files for subsequent processing and analysis. The exported spectra were pre-processed by an open-source software developed at the Vibrational Spectroscopy Core Facility in Umeå (https://www.umu.se/en/research/infrastructure/visp/downloads/), written in MATLAB (version 2014a-2018b, Mathworks, USA), using asymmetric least squares baseline correction (Eilers, 2004); (lambda: 100,000 and p=0.001), Savitzky-Golay smoothing (Savitzky and Golay, 1964); using a 1st order polynomial, with a frame number of 5; and total area normalization. Multivariate Curve Resolution—Alternating Least Squares (MCR-ALS) analysis was performed on the spectra using 5 components (based on singular value decomposition of the initial dataset). A maximum of 50 iterations and a convergence limit of 0.1 were used, with initial estimates in the spectrum direction (determined automatically by the built-in SIMPLISMA based algorithm) and a noise level of 10% given in the script. Only non-negativity constraints were used, both in the spectrum and concentration dimensions. The resulting profiles explained 99.84% of the variation. For classification, k-means clustering was performed within this open-source software, using the resolved spectral profiles and MATLAB's built-in algorithm.

[0124] Clubroot Infection Rating

[0125] All experiments were performed with the Plasmodiophora brassicae single-spore isolate e3 (Fähling et al., 2003, Journal of Phytopathology 151, 425-430) and Arabidopsis thaliana ecotype Columbia was used as the wild-type. Resting spores were extracted by the homogenization of mature clubroot galls (stored at −20° C.) of Chinese cabbage, followed by filtration through gauze (25-mm pore width) and two centrifugation steps (2,500×g, 10 min). Fourteen-day-old Arabidopsis seedlings, which were cultivated in a controlled environment (23° C., 16-h light, 100 mmol photons/s/m2) using a compost:sand (9:1 v/v) mixture (pH 5.8), were inoculated by injecting the soil around each plant with 1 ml of a resting spore suspension in Na.sub.2HPO.sub.4 buffer (pH 5.8), with the spore concentration 10.sup.6, 10.sup.5 and 10.sup.4 spores per ml. Controls were the same age and were treated with Na.sub.2HPO.sub.4 buffer (pH 5.8) (mock) instead of spore suspension. Disease symptoms were assessed at 28 days after inoculation (dai). At least 30 Arabidopsis plants were analyzed for each line and treatment. The disease severity was assessed qualitatively on the basis of the infection rate and a disease index (DI) as described by Siemens et al., (2002) using the following 5-scale classification: 0 (no symptoms), 1 (very small galls mainly on lateral roots and that do not impair the main root), 2 (small galls covering the main root and few lateral roots), 3 (medium to large galls, also including the main root; plant growth might be impaired), and 4 (severe galls on lateral root, main root, or rosette; fine roots completely destroyed; plant growth is reduced). Data are displayed as percentage of plants in the individual disease classes since this gives a more detailed view on the differences. Data presented are means of two independent experiments for the 10.sup.6 and 10.sup.5 spore concentrations and one for the 10.sup.4 spore concentration. The qualitative disease assessment data were analyzed using the Kruskal-Wallis-test and by subsequently comparing the mean rank differences as described in Siemens et al., (2002, Journal of Phytopathology 150, 592-605).

[0126] Accession Numbers

[0127] Sequence data relating to this invention can be found in GenBank/EMBL data libraries under the following accession numbers: SYAC1, At1 g15600; YIP5b, At3 g05280; YIP4a, At2 g18840; YIP4b At4 g30260; ECH, At1 g09330; KCR1, At1 g67730; DSK2, At2 g17200; PHB4, At3 g27280.

[0128] Results

[0129] Auxin and Cytokinin Synergistically Control Expression of SYAC1 in Root.

[0130] To search for novel molecular components and mechanisms of auxin-cytokinin cross-talk, genome wide transcriptome profiling of roots exposed to auxin, cytokinin and both hormones simultaneously, was performed. SYNERGISTIC AUXIN CYTOKININ 1 (SYAC1, AT1G15600), which encodes a protein of unknown function, was identified as a gene whose expression was synergistically up-regulated by simultaneous hormonal treatment when compared to the expected additive effect of both hormones applied separately. Whereas 3 hours of treatment with either auxin or cytokinin increased SYAC1 expression (2.47±0.20 and 1.53±0.19, respectively), application of both hormones simultaneously resulted in 16.36±0.14 higher expression compared to an untreated control. This SYAC1 expression profile in roots was further validated by quantitative real-time (RT-qPCR) (FIG. 2A). To examine the spatio-temporal pattern of SYAC1 expression and its responsiveness to hormones in roots, the SYAC1 promoter was cloned and used with GUS and nuclear localized GFP reporters. The basal expression of both pSYAC1: GUS and pSYAC1:nlsGFP reporters was under the threshold of detection, however exposure to cytokinin for 6 hours enhanced reporter signal in the quiescent center (QC) and columella initials (CI) (FIG. 2B). When treated with auxin the activity of pSYAC1 in the QC and provasculature of the root apical meristem could be detected (FIG. 2B). The promoter activity of pSYAC1 was substantially enhanced by the simultaneous application of both hormones, and remarkably, a strong reporter signal was detected in the differentiation and rapid elongation zone, a pattern not observed in roots exposed to either of the hormones separately (FIG. 2B). As initial concentrations of 1 μM auxin and 10 μM cytokinin for transcriptome profiling within 3 hours were relatively high, the sensitivity of the pSYAC1 reporter line to varying concentrations of both hormones was tested. This confirmed that pSYAC1 sensitively responds to simultaneous application of both hormones, and that their supply in concentrations of 0.25 μM each is sufficient to trigger reporter expression in the root differentiation and elongation zone. These results demonstrate that the SYAC1 promoter is under the tight control of the combined and synergistic action of auxin and cytokinin. When each hormone is applied individually, SYAC1 expression is activated in cells known to exhibit either auxin or cytokinin response maxima, such as QC/columella initials or cells of the provasculature, respectively. Intriguingly, SYAC1 transcription in the root differentiation and elongation zone is fully dependent on the simultaneous enhancement of both auxin and cytokinin.

[0131] As application of cytokinin and auxin might lead to deregulation of other hormonal pathways, in particularly that of ethylene, the sensitivity of SYAC1 to this hormone was also examined. No enhancement of pSYAC1:GUS expression was detected in roots treated with either 1-aminocyclopropane-1-carboxylic acid (ACC, a precursor of ethylene biosynthesis) only or in combination with either cytokinin, auxin or both hormones together. Taken together, the expression analysis confirms SYAC1 as a novel common target of the auxin and cytokinin pathways acting in roots.

[0132] SYAC1 Expression in Planta Spatio-Temporally Correlates with Reduced Cell Growth

[0133] To explore in which growth and developmental processes SYAC1 might be involved, its expression was monitored throughout the lifespan of Arabidopsis thaliana. Strong SYAC1 expression was detected in the embryonic hypocotyl and cotyledons, but not in the embryonic root (FIG. 2D). During germination, in 2-day-old seedlings, the pSYAC1:GUS activity remained strong in cotyledons and upper part of hypocotyl, however in the lower hypocotyl its expression ceased almost completely with commencing rapid elongation growth (FIG. 2E). As growth of hypocotyl and cotyledons progressed, in 3 to 4-day-old seedlings, SYAC1 expression in these organs gradually attenuated (FIG. 2F, G). In etiolated seedlings the SYAC1 expression was concentrated in short cells at the inner (concave) side of the apical hook, whereas no signal was detected in expanded cells at outer side of the hook (FIG. 2H). Based on these data, SYAC1 function seems not be limited to plant roots and its expression pattern spatio-temporally correlates with processes involving the control of elongation growth.

[0134] SYAC1 Regulates Elongation Growth of Plant Organs

[0135] To gain insights into the developmental function of SYAC1, detailed phenotypic analysis of plants with either a lower or an enhanced activity of this gene was performed. Characterization of the available mutant alleles revealed that the T-DNA is inserted either in the middle of the 3′ untranslated region (UTR) (syac1-1, syac1-2, syac1-3) or in the middle of the second intron (syac1-4), and thus is not fully suppressing SYAC1 expression. To obtain a syac1 knock-out line, we used the CRISPR/Cas9 approach was used. In the syac1-5 the CRISPR/Cas9 cassette introduces an extra thymine at 90 bps after the ATG, which results in a STOP codon after 33 amino acids in the SYAC1 coding sequence. Additionally, to investigate the impact of increased SYAC1 activity on plant development, the genetically modified lines SYAC1-HAox, HA-SYAC1ox, SYAC1-GFPox and GFP-SYAC1ox carrying SYAC1 fused to either the -HA tag or a GFP reporter under control of the 35S promoter were generated.

[0136] Given the observed pattern of SYAC1 expression, studies were focused on growth processes involving the control of cell expansion such as apical hook development, hypocotyl elongation, and primary root growth. Specific expression at the concave side of the apical hook prompted investigation into the SYAC1 function in this developmental process. In control Arabidopsis seedlings, shortly after germination (about 15-20 h), the hypocotyl progressively bent to establish an apical hook with an angle around 180° (formation phase). This bend was stabilized during the maintenance phase and subsequently, about 60 h after germination, a progressive opening of the hook occurred (opening phase). Overexpression of SYAC1 prevented formation of the apical hook bend, severely interfering with apical hook development. In contrast, in syac1-3 and syac1-5, the formation phase occurred at a similar rate to the wild-type control, but the maintenance phase was shortened and the opening of the hook started already 35 hours after germination. Introduction of pSYAC1:gSYAC1-GFP into the syac1-3 background rescued this defect and prolonged the maintenance phase until 60 h after germination as observed in wild-type seedlings. Apical hook development is the result of tightly orchestrated differential growth along the apical-basal axis of the hypocotyl. Since the SYAC1 expression maximum occurs in the shorter, concave side of the apical hook curvature (FIG. 2H), these data suggest that local accumulation of SYAC1 restricts expansion of cells locally at the inner side of hook and thereby coordinates the timely transition of the closed apical hook to the opening phase. Disruption of this endogenous expression pattern in SYAC1ox leads to inhibition of cell expansion on both sides of the hypocotyl which prevents formation of the apical hook. Hence, SYAC1 might play an important role in the regulation of differential growth, possibly by fine tuning cell elongation. Consistent with this notion, modulation of SYAC1 activity affected growth of hypocotyls. In 4-day-old dark-grown etiolated seedlings hypocotyls were significantly longer in both syac1-3 and syac1-5 alleles, whereas SYAC1 overexpression resulted in severe reduction of hypocotyl length when compared to the wild-type control. Since hypocotyl growth in darkness is largely driven by cell elongation rather than cell proliferation, the hypocotyl growth defects observed in syac1 mutants and SYAC1ox further support the SYAC1 function in regulation of cell elongation.

[0137] Close analysis of root growth did not reveal any significant alterations in syac1-3 and syac1-5 compared to the wild-type when grown on either control or hormone supplemented media. It was therefore tested whether SYAC1 might operate in root growth adaptation to transient hormonal fluctuations. 5-day-old syac1-3 and syac1-5 seedlings revealed significantly reduced sensitivity to transient increases of auxin and cytokinin when compared to wild-type. It is therefore hypothesized that under constitutive hormonal treatment conditions other proteins might compensate for the absence of SYAC1. An in silico search for SYAC1 related genes in the Arabidopsis genome identified a family of eight highly similar (40-60%) homologous genes of which seven are located as a cluster on chromosome 1. Among these, we found that BROTHER OF SYAC1 (BSYAC1), a close homologue of SYAC1, is also synergistically regulated by auxin and cytokinin, and thus presumably partially compensates for the loss of syac1 activity. By contrast, overexpression of SYAC1 significantly reduced root length when compared to wild type. Monitoring root growth revealed that estradiol-triggered induction of SYAC1 expression triggered a steep deceleration in root growth and indicating that SYAC1 effectively feeds back onto the kinetics of root elongation. In sum, these results suggest that SYAC1 acts as a developmentally specific regulator of elongation growth, whose activity is required to coordinate specific phases of apical hook development as well as the growth of other organs, such as hypocotyls and roots.

[0138] SYAC1 Localizes to the Secretory Pathway Compartments

[0139] To explore SYAC1's cellular function, its subcellular localization in Arabidopsis root cells was compared with specific reporters for cellular compartments. In the estradiol inducible line, 5 hours after induction SYAC1-GFP is restricted to small compartments in the cell interior. Measurement of Pearson correlation coefficient revealed a high SYAC1 co-localization pattern with Golgi and trans-Golgi (TGN) compartments labeled by the anti-SEC21 (0.57±0.01) and anti-ECH (0.51±0.02) antibody, respectively. This subcellular localization was further confirmed by anti-ARF1 (0.45±0.02) and anti-SYP61 (0.49±0.02) antibodies, which label both Golgi and TGN. A significant co-localization was also observed with the prevacuolar/endosomal compartments (PVC) labeled with a mixture of anti-ARA7 and anti-RHA1 (0.52±0.02) antibodies. In contrast, almost no co-localization was observed between SYAC1 and anti-BIP2 (0.04±0.04) and anti-PIN2 (0.03±0.04) antibodies, which label ER and plasma membrane, respectively. Accordingly, the SYAC1-GFP signal in SYAC1-GFPox line exhibited strong co-localization with markers for Golgi (anti-SEC21; 0.55±0.02), TGN (anti-ECH 0.60±0.02), and for both of them together (anti-ARF1; 0.55±0.02 and anti-SYP61; 0.40±0.02) and PVC (anti-ARA7/anti-RHA1; 0.44±0.02) but almost no co-localization with markers for ER (anti-BIP2; 0.01±0.03) and the plasma membrane (anti-PIN2; 0.02±0.02). To further validate the immunocolocalization results, the GFP-SYAC1ox line was crossed with the multicolor ‘Wave’ marker set (Geldner et al., 2009) for analysis of plant endomembrane compartments. This confirmed co-localization of SYAC1 with markers for Golgi (wave 18R; 0.53±0.03 and wave 127R; 0.42±0.02), Golgi and endosomes (wave 25R; 0.69±0.03 and wave 29R; 0.35±0.03), Golgi and TGN (SYP61:SYP61-CFP; 0.45±0.02), TGN and early endosomes (wave 13R; 0.27±0.06) as well as for endosomes/recycling endosomes (wave 34R; 0.31±0.05 and wave 129R; 0.33±0.02). In agreement with immunocolocalization, SYAC1 displayed only minor co-localization with markers for ER/plasma membrane (wave 6R; 0.06±0.02), plasma membrane (wave 131R; 0.02±0.02 and wave 138R; 0.02±0.03) and vacuoles (wave 9R; 0.03±0.02). These results strongly support that SYAC1 largely resides in the Golgi, TGN, and Endosomal and PVC compartments.

[0140] SYAC1 is a Novel Component of the ECHIDNA/Yip Complex

[0141] To further assess SYAC1's molecular function, its molecular interactors were identified using a tandem affinity purification assay with SYAC1 as bait. Proteins including the integral membrane YIP1 family protein (YIP5b), β-ketoacyl reductase 1 (KCR1), an ubiquitin receptor protein (DSK2), and prohibitin 4 (PHB4) were recovered by this approach. As YIP5b is a member of the YIP (for YPT/RAB GTPase Interacting Protein) family in Arabidopsis thaliana that forms a TGN-localized complex with YIP4a and YIP4b homologues and Echidna (ECH) integral membrane protein, they were included them in subsequent detailed interaction studies. A Yeast two-hybrid assay (Y2H) revealed a strong interaction between SYAC1 and all three YIP family members. Moreover, SYAC1 interacted well with ECH, but only weakly with KCR1 and not at all with the DSK2 and PHB4 proteins. The Y2H results were further validated in planta using a bimolecular fluorescence complementation (BiFC) assay. SYAC1 tagged with the C-terminus of EYFP, and YIP5b, YIP4a, YIP4b, ECH, KCR1, DSK2 and PHB4 tagged with the N-terminus of EYFP, were transiently expressed in an Arabidopsis root suspension culture. Yellow fluorescence was detected in protoplasts overexpressing SYAC1 in combination with YIP5b, YIP4a, YIP4b and ECH, indicating the close physical proximity of these proteins in vivo. By contrast, no EYFP reconstitution was detected in cells overexpressing SYAC1 with KCR1, DSK and PHB4, respectively, in agreement with the result of the Y2H assay. Finally, the interaction between SYAC1 and YIP4a and between SYAC1 and ECH was also confirmed by a co-immunoprecipitation (Co-IP) assay. Results from tandem affinity purification, BiFC and Co-IP assays revealed SYAC1 interaction with YIP5b, YIP4a, YIP4b and ECH protein, and indicate its function in the protein complex involved in maintaining the functionality of the secretory pathway.

[0142] SYAC1 Determines Secretory Pathway Activity

[0143] SYAC1 localization in Golgi/TGN/Endosomal/PVC compartments and the interaction with ECH/YIPs pointed towards a potential function in the secretory pathway. The secretory pathway is of vital importance for all eukaryotic cells, since it manufactures, stores and distributes macromolecules, lipids and proteins as cargo to intracellular and extracellular locations. To assess the involvement of SYAC1 in the regulation of secretion, transient expression assays were performed in Arabidopsis mesophyll protoplasts and the impact assessed of SYAC1-HAox or HA-SYAC1ox on the secretory index of the α-Amylase (Amy) reporter—a protein that without any intrinsic sorting signal is secreted extracellularly and can be detected by its endogenous enzymatic activity. The secretion index was determined by quantifying the ratio of the α-Amylase activity in the medium and in the cells. Expression of the SYAC1 protein decreased the secretion index from 0.70±0.04 in control sample to 0.55±0.02 (SYAC1-HAox) and 0.45±0.01 (HA-SYAC1ox), which suggests a function of SYAC1 as a negative regulator of the anterograde secretory route to the cell surface. Because of SYAC1's co-localization with markers for PVC compartments, SYAC1's involvement in transport to the vacuoles was investigated. For that, an α-Amylase with a vacuolar sorting signal (Amy-Spo) was co-transfected with either SYAC1-HA or HA-SYAC1 encoding plasmids. The secretion index of α-Amylase (Amy-Spo) was increased from 0.07±0.01 in the control sample to 0.29±0.01 (SYAC1-HAox) and 0.28±0.03 (HA-SYAC1ox), which suggests that SYAC1 impairs transport to vacuoles leading to more α-Amylase secretion out of the cells. SYAC1's effect on α-Amylase containing an ER retention signal (Amy-HDEL), which redirects the protein back to the ER was tested. Co-transfection of SYAC1 significantly decreased the secretion index in protoplasts with leaky retention of α-Amylase from 0.34±0.01 in the control sample to 0.24±0.01 (SYAC1-HAox) and 0.26±0.04 (HA-SYAC1ox). Altogether these results support the conclusion that SYAC1 modulates the activity of the secretory pathway, and coordinates cargo trafficking towards the extracellular space and vacuoles.

[0144] The Effect of SYAC1 Expression on Plant Cell Wall Composition

[0145] In plants, new cell wall components such as pectins and hemicellulose are proposed to be delivered to the extracellular matrix via the secretory pathway (reviewed in Wolf and Greiner, 2012, Protoplasma 249, 169-175). SYAC1 reduction of α-Amylase secretion, along with its Golgi/TGN/Endosomal localization and interaction with YIPs and Echidna proteins suggested a role for SYAC1 in the control of soluble cell wall polysaccharides (pectin and hemicellulose) secretion. Investigating the seed coat epidermis, whose TGN is highly specialized for pectic mucilage secretion using ruthenium red staining assay revealed that mucilage release from mature seeds was greatly reduced in SYAC1-GFPox seeds, relative to wild-type (FIG. 7), which is in line with the anticipated function of SYAC1 as an inhibitor of polysaccharide secretion and with the previously described ech-1 and yip4ayip4b mutants.

[0146] Delivery of new cell wall components during pollen tube growth is a particularly active process. The effects of SYAC1 expression on pectin secretion in tobacco pollen tubes was studied and it was observed that SYAC1 severely affected accumulation of pectin at the pollen tip, supporting its role as a factor modulating the activity of the secretory pathway (FIG. 10). As in Arabidopsis root cells, the localization of plasma membrane proteins such as PIN1 and PIN2 was not affected, suggesting that SYAC1 might preferentially regulate specific branches of the secretory pathway. Taken together, these data support SYAC1 function in modulation of the cell wall matrix polysaccharides delivery.

[0147] To assess the impact of the modulated activity of SYAC1 on cell wall composition, hypocotyls of etiolated seedlings were inspected using Fourier transform-infrared (FT-IR) microspectroscopy. FT-IR analysis revealed that enhanced SYAC1 activity in plant cells substantially alters the composition of cell walls, which is manifested by a significantly reduced proportion of carbohydrates (FIG. 3). To further dissect the qualitative changes in the cell wall, analyses of pectin content and xyloglucans, two major components of cell walls, in hypocotyls of seedlings with modified SYAC1 expression were performed. A quantitative analysis of galacturonic acid, a key structural component of pectin, did not reveal any significant changes in hypocotyls of the syac1 loss of function mutant when compared to the control, consistent with the relatively modest defects on syac1 hypocotyl growth. By contrast, the amount of galacturonic acid extracted from hypocotyls of seedlings with enhanced expression of SYAC1 was significantly reduced when compared to control (FIG. 5C). The pectic polysaccharides are secreted in a methylesterified form. In correlation with a reduced amount of pectin in the cell wall, methanol content was decreased in hypocotyls of seedlings with enhanced SYAC1 activity. Unlike pectin, no changes in the amount of fucose, galactose and xylose which are indicators of xyloglucan content in the cell wall could be detected in seedlings with either loss or enhanced activity of SYAC1. Altogether these results suggest that SYAC1 interferes with a specific branch of the secretory pathway mediating pectin delivery to the cell wall.

[0148] Secretion defects which lead to alterations in cell wall composition might ultimately result in changes in cell wall physical properties, and on plant wall stiffness in particular. Analyses of etiolated hypocotyls using atomic force microscopy (AFM) revealed a significantly reduced apparent Young modulus on the extracellular matrix upon enhanced SYAC1 activity when compared to control (FIG. 4). Altogether, these results support the conclusion that SYAC1 acts as a regulator of the TGN-mediated secretion of cell wall components such as pectins and ultimately affects the composition and physical properties of cell walls.

[0149] The results presented in FIG. 4, which compares a control plant: Arabidopsis thaliana (L.) Heynh ecopyte Columbia and with a genetically modified plant according to the invention: SYAC1-GFPox, confirm a difference in stiffness between the cell walls on control plants and the cell walls of SYAC1 overexpressor (SYAC1-GFPox) plants. This reduced stiffness of the cell walls in the genetically modified plants increases the efficiency of cell wall-degrading enzymes and of the extraction of polysaccharides.

[0150] SYAC1 as a Spatio-Temporal Modulator of YIP/ECH Complex Function

[0151] SYAC1 was shown to interact with YIPs and ECH, components of the protein complex required for the proper operation of the secretory pathway. Intriguingly, compromised functionality of the YIP/ECH complex leads to cellular and developmental defects reminiscent of these caused by enhanced activity of SYAC1. Similarly to SYAC1ox, deficiency in the secretion of pectins, as well as root, hypocotyl and apical hook development defects have been observed in yip4a, yip4b and ech loss of function mutants. To explore SYAC1 function as a potential attenuator of YIP/ECH complex activity, tests were carried out to determine whether relief of the SYAC1 mediated inhibition of the secretory pathway might alleviate growth defects caused by defects of the YIP/ECH complex. To test this hypothesis, the syac1-3 allele was introduced into the yip4a, yip4b mutant background, and the resulting combined genotype was phenotyped. Importantly, the syac1-3 yip4a yip4b triple mutant displayed significantly improved growth of hypocotyls and shoot organs when compared to the yip4a, yip4b double mutant, indicating that SYAC1 might indeed act as a negative regulator of the YIPs/ECH complex. Based on these observations it is hypothesized that whereas constitutively expressed YIPs and ECH act as generic factors required for the secretion of cell wall components to maintain cell expansion, the spatio-temporally controlled expression pattern of SYAC1 may allow it to act as a developmentally specific regulator of the YIP/ECH complex to fine tune secretory pathway activity and thereby plant organ growth.

[0152] SYAC1 Expression Results in an Increase in Hemicellulose Expression in Plant Cell Walls

[0153] In order to demonstrate that SYAC1 expression results in an increase in hemicellulose expression in plant cell walls, xyloglucan levels were detected using a labelled antibody. FIG. 5A and FIG. 5B demonstrate, using immunolacalization of xyloglucan with an LM 15 antibody, an increase and change in xyloglucan localization in SYAC1-GFPox line. The immunolabeling was performed as described above.

[0154] SYAC1 Expression Results in Reduction in Galacturonic Acid in Plant Cell Walls

[0155] FIG. 5C demonstrates that when the expression of SYAC1 is increased, the amount of galacturonic acid in the plant cell wall decreases. Analysis of the cell wall was performed on 4 days old dark grown hypocotyls as described above.

[0156] SYAC1 Expression Affects the α-Amylase Secretion Index

[0157] α-Amylase assays and calculations of the secretion index were performed as described by Früholz and Pimpl (2017) in Plant Protein Secretion, (Humana Press, New York, N.Y.), pp. 171-182. α-Amylase expression constructs were kindly provided by P. Pimpl and transfections were performed in Arabidopsis mesophyll protoplasts. α-Amylase activity was measured with the Ceralpha kit from Megazyme. The reaction was performed in a microtiter plate at 37° C. with 30 μL of extract and 30 μL of substrate. The reaction was stopped by the addition of 150 μL of stop buffer. The absorbance was measured at a wavelength of 405 nm. Experiment was performed three times with four replicates.

[0158] These results shown in FIG. 6 indicate that SYAC1 modulates activity of a secretory pathway, and coordinates trafficking of cargos towards extracellular space in plant cells. The changes observed in the cell wall modification in SYAC1ox lines are caused by regulation of secretory pathway. This mechanism clearly distinguishes SYAC1 from other genes known to regulate cell wall composition. Results shown in FIGS. 13A-C indicate similar regulation of secretory pathway also by SYAC1 paralogues.

[0159] SYAC1 Expression Inhibits Seed Coat Mucilage Secretion

[0160] In order to demonstrate that SYAC1 expression inhibits seed coat mucilage secretion, mature seeds of Arabidopsis thaliana (L.) Heynh ecopyte Columbia and SYAC1-GFPox were incubated in 0.01% (w/v) aqueous solution of Ruthenium red. Seeds were mounted in water and viewed using a DIC Olympus BX53 microscope.

[0161] The results in FIG. 7 show that mucilage release from mature seeds is significantly reduced in SYAC1-GFPox seeds, relative to Columbia control cells. The result confirms that SYAC1 plays a role in the regulation of cell wall components secretion.

[0162] SYAC1 Expression Improves Pathogen Defence

[0163] To examine whether SYAC1 is useful in host-pathogen interaction and defence the sensitivity of plants with altered SYAC1 expression to the pathogen Plasmodiophora brassicae was studied. The analysis of root and shoot phenotypes after inoculation with the plant pathogenic protist was done at three different spore concentrations (10.sup.6, 10.sup.5 and 10.sup.4 spores mL-1). While a high inoculation pressure should identify tolerant plants, low spore concentrations will reveal oversensitive phenotypes. To characterize disease progression 5 categories was used (Siemens et al., 2002, Journal of Phytopathology 150, 592-605): zero denotes no infection, 1 almost no infection, 2 and 3 intermediate infection phenotypes and 4 a root completely transformed into a clubroot. Both syac1-3 and syac1-5 mutant alleles exhibited increased tolerance to the pathogen when compared to the wild-type control, unlike plants overexpressing SYAC1 which exhibited oversensitivity to pathogen infection (FIGS. 11 and 12). Both root and shoot phenotypes of infected plants are in line with these observations. In both mutant plant lines more roots were classified into lower disease classes at high and medium spore concentrations than in wild-type while at the low spore concentration no differences were observed (FIG. 11). On the contrary, all SYAC1 overexpressors showed more roots in class 4 when compared to the wild-type at the lowest spore concentration indicating their oversensitivity to the pathogen. Rosette development followed the same pattern (FIG. 12). Altogether these results indicate that SYAC1-mediated control of cell wall composition in roots is of crucial importance for root-pathogen interaction and the suppression of SYAC1 expression in roots could be the result of evolutionary selection to restrain invasions of pathogens through the root system.

[0164] The results demonstrated that by reducing the expression of SYAC1 in a plant, such as a a Brassica species, susceptibility to some pathogenic organisms, such as fungi, can be reduced. In particular the susceptibility of Brassica species to diseases caused by Plasmodiophora brassicae, such as club root disease can be reduced. A reduction in susceptibility to a pathogenic organism may result in a reduced ability of the organism to colonise the plant.

[0165] The data presented herein demonstrates that where plants were infected with 10.sup.5 spore concentrations of P. brassicae, almost 60% of syac1 mutant lines showed reduced susceptibility to the pathogen in comparison to only 30% of wild-type plants. With increased spore concentration (10.sup.6) still almost 40% of mutant lines maintained resistance, compared to only 15% in wild-type plants. In addition to clubroot resistance, earlier flowering was also observed in syac1 mutant lines which could potentially be of interest for reducing time needed to finish the life cycle. On the contrary, all SYAC1 overexpressors showed increased sensitivity to pathogen compared to the wild-type already at the lowest spore concentration.

[0166] SYAC1 Expression Renders Plant Cells More Tolerant to Copper

[0167] FIG. 8 shows the effect of excess copper on the primary root growth of seedlings grown on agar. Plants were grown for 5 days on normal AM+ media and then transferred to media containing 50 μM CuSO.sub.4. CuSO.sub.4 is shown to trigger swelling of cells at the root tip of wild type control plants, but not SYAC1-GFPox plants. These results demonstrate that SYAC1 expression renders plant cells more tolerant to elevated copper.

[0168] FIG. 9 further supports the ability of SYAC1 expression to render plants suitable for cultivation on contaminated soil. In particular, the results demonstrate that SYAC1 expression significantly reduces the amount of Cu or Fe taken up by plant roots. Wild type Col-0 and the SYAC1 overexpressor lines (HA-SYAC1ox) were grown for 1 week in standard MS media on agar plates. Fresh roots were collected and rinsed in deionized water during the sampling and stored in Falcon tubes. To remove the copper in the cell apoplast, fresh roots samples were immediately washed once in 40 ml 1 mM HCl solution (shaking end over end) for 3 mins. First washing solution was removed and samples were additionally washed in 0.01M HCl for 5 mins. Finally, root samples were rinsed in 40 ml deionized H.sub.2O (Chaignon and Hinsinger, J. Environ. Qual., 2003). Samples were dried overnight in Falcon tubes at 65° C. in oven. For analysis of metal content, sample digestion has been performed at 150° C. for 4 h in a mixture of 65% HNO.sub.3 and 30% H.sub.2O.sub.2 in a ratio of 5:1 (v/v). Copper and iron were measured by detecting two isotopes, Cu-63 and Cu-65 or Fe-54 and Fe-57 respectively in a Perkin Elmer Elan DRCe 9000 (Rosenkranz et al., Waste Manag., 2018).

[0169] SYAC1 Expression in Tobacco Pollens Tubes Alters Pectin Secretion

[0170] Mature pollen was collected from four to six tobacco (Nicotiana tabacum) flowers of 8-week-old plants. Pollen was resuspended in growth medium, filtered onto cellulose acetate filters, and transferred to Whatman paper moistened with growth medium. Within 5 to 10 min of harvesting, pollen was transformed by bombardment with plasmidcoated 1-mm gold particles with a helium-driven particle accelerator (PDS-1000/He; Bio-Rad) using 1350 p.s.i. rupture discs and a vacuum of 28 inches of mercury. Gold particles (1.25 mg) were coated with 3 to 7 mg of plasmid DNA. After bombardment, pollen was resuspended in growth medium and grown for 5 to 14 h in small droplets of media directly on microscope slides. Digital images were taken under the light microscope (Olympus BX51) within 5 to 15 min after addition of the dye. Pollen tubes transformed with eYFP (enhanced Yellow fluorescent protein) were taken as control to pollen tubes transformed with SYAC1 tagged with mCherry protein)

[0171] The data presented in FIGS. 10A and 10B clearly show that the expression of SYAC1 in the pollen tubes results in a change in the normal pattern of pectin secretion, when compared to a control pollen tube which does not express SYAC1.

CONCLUSION

[0172] The results presented here demonstrate that SYAC1 expression results in a reduction of α-amylase secretion, along with the observation that it localizes in the golgi/TGN/endosomes and interacts with YIPs and Echidna proteins, this demonstrates a role for SYAC1 in the control of soluble cell wall polysaccharides (pectin and hemicellulose) secretion. Investigating the seed coat epidermis, in which the TGN is highly specialized for pectic mucilage secretion using ruthenium red staining assay revealed that mucilage release from mature seeds was greatly reduced in SYAC1-GFPox seeds, relative to Columbia (FIG. 7) which is in line with a function of SYAC1 as a regulator of polysaccharide secretion. Hemicellulose components of cell wall, monitored using LM15 an anti-xyloglucan antibody, were enriched around QC (quiescent centre) in Columbia control roots. In the SYAC1-GFPox line, enhanced staining of root epidermal, cortex and endodermal was detected. Taken together, this data supports a role for SYAC1 in the modulation of cell wall matrix polysaccharides delivery, including both hemicelluloses and pectins. The defects in the secretion of cell wall components leads to alterations in the cell wall structure and its physical properties. To assess whether SYAC1 triggered changes in delivery of cell wall components affect the composition and physical properties of cell walls, hypocotyls of etiolated seedlings were inspected using Fourier transform-infrared (FT-IR) microspectroscopy and atomic force microscopy (AFM). FT-IR analysis revealed that enhanced SYAC1 activity in plant cells substantially alters the composition of the cell walls, which is manifested by a significantly reduced proportion of carbohydrates (FIG. 3). Accordingly, an increase in SYAC1 activity has a direct impact on the physical properties of cell walls and reduces their stiffness (FIG. 4). Thus, these results indicate that SYAC1 is an essential regulator of the TGN-mediated secretion of cell wall components such as pectins and xyloglucan and ultimately affects the composition and physical properties of cell walls.

[0173] The efficient conversion of lignocellulosic biomass into fuel ethanol has become a world priority for producing environmentally friendly renewable energy at a reasonable price for the transportation sector. The main source of lignocellulose is plant secondary cell walls, this is the thick, strengthening layer of the cell wall that is laid down inside the primary wall after cell elongation has terminated. Approximately 75% of lignocellulose is comprised of polysaccharides, which can potentially be converted into monosaccharides for fermentation. The main constituents of plant secondary cell walls are cellulose, hemicellulose and lignin, and these are present in varying proportions in different feedstocks. The cellulose fibres are embedded in a matrix of hemicellulose and, in primary cell walls, pectin, which appear to play a dual role of strengthening and increasing extensibility of the wall to enable cell enlargement. In secondary cell walls, the polysaccharide network is impregnated and coated by lignin, which provides rigidity and strength. Lignin strengthens the cell wall, increasing its hydrophobicity and posing a formidable barrier to cell wall-degrading enzymes. Plant cell wall softening caused by SYAC1 overexpression, and demonstrated herein, leads to a decrease of hydrophobicity and opens the way for degrading enzymes. Similarly, an increase in hemicellulose content provides new material for sugar extraction. Thus plants or plant cells with increased SYAC1 levels are a good source of readily accessible polysaccharides for use as biomass in energy production.