The mutualism effector MiSSP7 of Laccaria bicolor alters the interactions between the poplar JAZ6 protein and its associated proteins

Abstract

Despite the pivotal role of jasmonic acid in the outcome of plant-microorganism interactions, JA-signaling components in roots of perennial trees like western balsam poplar (Populus trichocarpa) are poorly characterized. Here we decipher the poplar-root JA-perception complex centered on PtJAZ6, a co-repressor of JA-signaling targeted by the effector protein MiSSP7 from the ectomycorrhizal basidiomycete Laccaria bicolor during symbiotic development. Through protein-protein interaction studies in yeast we determined the poplar root proteins interacting with PtJAZ6. Moreover, we assessed via yeast triple-hybrid how the mutualistic effector MiSSP7 reshapes the association between PtJAZ6 and its partner proteins. In the absence of the symbiotic effector, PtJAZ6 interacts with the transcription factors PtMYC2s and PtJAM1.1. In addition, PtJAZ6 interacts with it-self and with other Populus JAZ proteins. Finally, MiSSP7 strengthens the binding of PtJAZ6 to PtMYC2.1 and antagonizes PtJAZ6 homo-/heterodimerization. We conclude that a symbiotic effector secreted by a mutualistic fungus may promote the symbiotic interaction through altered dynamics of a JA-signaling-associated protein-protein interaction network, maintaining the repression of PtMYC2.1-regulated genes.

Figure 1

PtJAZ6 interacts with PtJAM1 and PtMYC2 transcription factors in yeast and in planta. (a) Phylogenetic analysis of the bHLH transcription factors orthologous to Arabidopis thaliana MYCs and JAMs. The evolutionary history was inferred through the Maximum Likelihood method and conducted in MEGA7. The values of the 1000 performed bootstraps are indicated. FigTree v1.4.4 (https://tree.bio.ed.ac.uk/software/figtree/) was used to draw the tree. (b) Yeast-two hybrid (Y2H) assays to reveal interactions of PtJAZ6 with PtJAM1.1, PtJAM1.2, PtMYC2.1 or PtMYC2.2. PtJAZ6 was fused to the GAL4 DNA-binding domain (DBD) whereas the various transcription factors (TFs) were fused to the GAL4-activation domain (AD). TRP1 and LEU2 served as transformation markers for vectors whereas HIS3, URA3 and β-galactosidase LacZ (X-Gal) were used as reporter genes. (c) Live-cell imaging of PtJAZ6:GFP and TF:RFP transiently expressed in Nicotiana benthamiana epidermal leaf cells transformed by agroinfiltration, using a laser-scanning confocal microscope in a sequential scanning mode. Scale bar: 20 μm. (d) In planta co-immunoprecipitation of PtJAZ6 with PtJAM1 or PtMYC2 proteins. Fusion proteins (PtJAZ6:GFP and TF:RFP) were isolated from agroinfiltrated leaves. Both initial proteins (Input) and immunoprecipitated protein (IP-GFP) were separated by 12% SDS-PAGE and electro-transferred onto PVDF membranes. Immunodetection was performed with anti-GFP or anti-RFP antibodies, and immunoblots were revealed with a chemiluminescent imager. Ponceau staining of the PVDF membrane was used as a loading control. White asterisks indicate specific protein bands. Numbers on the left side of the blots indicate protein size in kilodaltons. (e) Y2H assays to assess which domains of PtJAZ6 are required for its interaction with PtMYC2.1 and PtJAM1.1. PtMYC2.1 and PtJAM1.1 were fused to the GAL4 AD, whereas full-length or mutated versions of PtJAZ6 were fused with the GAL4 DBD. TRP1 and LEU2 served as transformation markers for vectors whereas HIS3 and β-galactosidase LacZ (X-Gal) were used as reporter genes. For Y2H (b and e): 3-Amino-1, 2, 4-triazole (3-AT) was used to suppress self-activation of the HIS3 gene. The control colonies are shown in the upper part of the panel: ++ is a strong positive interaction, + is a weakly interacting protein pair, — is a negative control (no interaction). Black and white lines indicate cropped and repositioned images while the full-length blots and gels are presented in Supplementary Information online.

Figure 2

PtJAZ6 can form homodimers and heterodimers with PtJAZ proteins through its TIFY domain. (a) Phylogenetic tree of canonical JAZ proteins from Arabidopsis and Populus. The evolutionary history was inferred through the Maximum Likelihood method and conducted in MEGA7. The values of the 1000 performed bootstraps are indicated. *Indicate PtJAZ proteins interacting with PtJAZ6 forming homo- and heterodimers. FigTree v1.4.4 (https://tree.bio.ed.ac.uk/software/figtree/) was used to draw the tree. (b) Yeast-two hybrid (Y2H) assays to detect interactions of PtJAZ6 with itself and with other PtJAZ proteins expressed in ECM root tips. PtJAZ proteins were fused to the GAL4 DBD. PtJAZ6 or PtJAZ6 deleted for its TIFY domain (PtJAZ6ΔTIFY) or containing the TIFY domain only (PtJAZ6 TIFY) were fused with the GAL4 AD. TRP1 and LEU2 served as transformation markers for vectors whereas HIS3 and β-galactosidase LacZ (X-Gal) were used as reporter genes. The control colonies are shown in the upper part of the panel: ++ is a strong positive interaction, + is a weakly interacting pair, – is a negative control (no interaction). 3-Amino-1, 2, 4-triazole (3-AT) was used to suppress self-activation of the HIS3 gene. White lines indicate cropped and repositioned images while the full-length blots and gels are presented in Supplementary Information online. (c) Quantification of β-galactosidase activity in yeast colonies showed in (b) through ONPG assay. ++ is a strong positive interaction, + is a weakly interacting pair, — is a negative control (no interaction). Significant differences between the interaction of PtJAZ proteins and the different deleted versions of PtJAZ6 were assessed via pairwise Student’s t-test (*p < 0.05, **p < 0.01).

Figure 3

PtJAZ6 interacts with one PtTOPLESS-related protein and two NINJA proteins. (a) Phylogenetic tree of Populus trichocarpa TOPLESS-related proteins expressed in poplar roots. The evolutionary history was inferred by using the Maximum Likelihood method and conducted in MEGA7. The values of the 1000 performed bootstraps are indicated. The three poplar TOPLESS-related proteins cloned from a poplar ECM cDNA library are indicated as PtTPR1.1, PtTPR4.1, and PtTPR4.2. FigTree v1.4.4 was used to draw the tree. (b) Yeast-two hybrid (Y2H) assays to reveal interactions of PtJAZ6 with the TOPLESS-related protein PtTPR4.1 expressed in ECM root tips. PtJAZ6 was fused to GAL4 AD whereas the three TOPLESS-related proteins were fused to the GAL4 DBD. (c) The C-terminus EAR motif of PtJAZ6 is required for its interaction with PtTPR4.1 in yeasts. Yeast-two hybrid (Y2H) assays were performed with yeast co-expressing DBD:PtTPR4.1 and AD:PtJAZ6 or AD:PtJAZ6 mutated for its EAR motif (EARm). (d) PtJAZ6 interacts with PtNINJA1 and PtNINJ3 and the TIFY domain of PtJAZ6 is necessary and sufficient for the interaction. Y2H assays were performed with yeast co-expressing DBD:PtNINJA1 or DBD:PtNINJA3 and full length or mutated version of PtJAZ6 were fused with the GAL4 AD. For all yeast-two hybrid assays (b,c,d), TRP1 and LEU2 served as transformation markers for vectors and HIS3 and β-galactosidase LacZ (X-Gal) were used as reporter genes. 3-Amino-1, 2, 4-triazole (3-AT) was used to suppress self-activation of the HIS3 gene. The control colonies are shown in the upper part of the panel: ++ is a strong positive interaction, + is a weakly interacting pair, — is a negative control (no interaction). White lines indicate cropped and repositioned images while the full-length blots and gels are presented in Supplementary Information online.

Figure 4

Both NT and TIFY domains of PtJAZ6 are necessary for its interaction with MiSSP7. (a) Yeast-two hybrid (Y2H) assays to assess which domains of PtJAZ6 are required for its interaction with MiSSP7. MiSSP7 was fused to the GAL4 DBD, whereas full length or mutated versions of PtJAZ6 were fused with the GAL4 AD. (b) Yeast-three hybrid assay to assess if the presence of MiSSP7 impacts the interaction between PtJAZ6 and PtJAZ6-PtCOI1. PtJAZ6 was fused to the GAL4 AD and PtCOI1 was fused to GAL4 DBD. Coronatine (COR) triggers the interaction betwen PtJAZ6 and PtCOI1. TRP1, LEU2 and URA3 served as transformation markers (the latter in case of triple hybrids) for vectors whereas HIS3 and β-galactosidase LacZ (X-Gal) were used as reporter genes. 3-Amino-1, 2, 4-triazole (3-AT) was used to suppress self-activation of the HIS3 gene. The control colonies are shown in the upper part of the panel: ++ is a strong positive interaction, + is a weakly interacting pair,— is a negative control (no interaction). 3-Amino-1, 2, 4-triazole (3-AT) was used to suppress self-activation at the HIS3 gene. White lines indicate cropped and repositioned images while the full-length blots and gels are presented in Supplementary Information online.

Figure 5

MiSSP7 impacts the structure of the jasmonate perception complex. (a, b) MiSSP7 differentially impacts the interaction between PtJAZ6 and the two isoforms of PtMYC2 but does not alter PtJAZ6-PtJAM1.2 or PtJAZ6-PtTPR4.1 interactions. PtJAZ6 was fused to the GAL4 DBD. PtMYC2s, PtJAM1.2, PtNINJA3 and PtTPR4.1 were fused with the GAL4 AD. (c, d) MiSSP7 antagonizes dimerization between PtJAZ6 and other PtJAZ proteins. PtJAZ6 was fused to the GAL4 AD and PtJAZ proteins were fused with the GAL4 DBD. For a) and c), yeasts were transformed with MiSSP7 to give triple hybrids. TRP1, LEU2 and URA3 served as transformation markers (the latter in case of triple hybrid colonies) while HIS3 and β-galactosidase LacZ (X-Gal) were used as reporter genes. 3-Amino-1, 2, 4-triazole (3-AT) was used to suppress self-activation of the HIS3 gene. The control colonies are shown in the upper part of the panel: + is a weakly interacting pair. For b) and d), the enzymatic activity of the β-galactosidase in different yeast colonies was quantified via ONPG assay. Significant differences between yeast colonies transformed or not with MiSSP7 were assessed via pairwise Student’s t-test (*p < 0.05, **p < 0.01). White lines indicate cropped and repositioned images while the full-length blots and gels are presented in Supplementary Information online.

Figure 6

PtJAZ6-interacting proteins in Populus ectomycorrhizal root tips. (a) PtJAZ6 physically interacts with the transcription factors PtMYC2s and PtJAM1s, as well as with PtTPR4.1, NINJA1 and NINJA3. It also forms homodimers and heterodimers with PtJAZ5, PtJAZ10.2 and PtJAZ12. PtJAZ6 probably suppresses the transcriptional activity of PtMYC2s and PtJAM1s by recruiting the general repressor PtTPR4.1 to the promoter of MYC2-responsive genes. (b) The fungal symbiotic effector MiSSP7 interacts with PtJAZ6 inhibiting homo- and heterodimerization of PtJAZ6. Such inhibition may alter the stability of PtJAZ6. The presence of MiSSP7 does not impair the interaction between PtJAZ6 and PtNINJA1 or PtNINJA3 However, MiSSP7 loosens the interaction between PtJAZ6 and PtMYC2.2, while strengthening the interaction between PtJAZ6 and PtMYC2.1. MiSSP7-mediated enhancement of the PtJAZ6-PtMYC2.1 interaction probably dampens the transcriptional activity of PtMYC2.1. On the other hand, the release of PtMYC2.2 from PtJAZ6-mediated repression may activate gene expression depending on this transcription factor.