Article Figures & Data
Figures
- Figure 1.
NSP1 and NSP2 Form Heteropolymers in Yeast.
(A) NSP1 and NSP2 Gal4 DNA binding domain fusions autoactivate the Gal4 system in S. cerevisiae. S. cerevisiae was grown on media with selection for growth and β-galactosidase activity. The N-terminal domains of NSP1 (1-155) and NSP2 (1-112) are sufficient for autoactivation, and if this domain is removed from NSP1 (NSP1 156-554), no autoactivation was observed. Autoactivation of NSP2 is abolished by the NSP2E232K mutation. NSP2E232K fused to the Gal4 DNA binding domain interacts with NSP1 fused to the activation domain.
(B) Quantification of β-galactosidase activity in S. cerevisiae revealed interactions between wild-type NSP1 and NSP2. One unit of β-galactosidase is defined as the amount that hydrolyzes 1 mmol o-nitrophenyl β-D-galactopyranoside (ONPG) per minute. Each value is the mean of three technical and three biological replicates. Error bars indicate the se of the three biological replicates. AD, Gal4 activation domain; BD, Gal4 DNA binding domain.
(C) The GRAS proteins are defined by two leucine heptad repeats (LHRI and LHRII) and three domains with highly conserved residues (VHIID, PYFRE, and SAW). Several N- and C-terminal deletions of NSP1 and NSP2 were fused to the Gal4 DNA binding domain and tested for interaction with NSP2 or NSP1, respectively. β-Galactosidase activity is shown. Please note the change in scale. Positive interactions are indicated with +, and − indicates no interaction.
- Figure 2.
NSP1 and NSP2 Form Heteropolymers in Planta.
(A) to (C) N. benthamiana leaves were cotransformed with YFPN-NSP1 and YFPC-NSP2 (A), YFPN-NSP1 and YFPC-NSP2-LHRI (B), and YFPN-NSP1 and YFPC-NSP2ΔLHRI (C). Overlays of confocal YFP and bright-field images of epidermal N. benthamiana leaf cells are shown. (A) and (B) show the nuclear localization of the NSP1 and NSP2 interaction. Removal of the LHRI domain of NSP2 abolished the interaction with NSP1 (C). The inset of (C) confirms protein stability of (a) YFPC-NSP2 (65 kD) and (b) YFPC-NSP2ΔLHRI (58 kD) in these N. benthamiana transient expression studies. Bars = 40 μm.
(D) NSP1-3xHA alone and NSP2-FLAG with NSP1-3xHA were transiently expressed in N. benthamiana leaves. Leaves were harvested 3 d after infiltration, and equal amounts of total protein extracts (input) were subjected to immunoprecipitation with α-FLAG antibodies. Analysis of the elution revealed coimmunoprecipitation of NSP1-3xHA with NSP2-FLAG that was not observed in the leaves transformed with NSP1-3xHA alone.
- Figure 3.
NSP1 and NSP2 Form Homopolymers.
(A) NSP1-3xHA alone or NSP1-FLAG and NSP1-3xHA were transiently expressed under the control of the 35S promoter in N. benthamiana leaves. Leaves were harvested 3 d after infiltration and NSP1-FLAG immunoprecipitated (elution) from total protein extracts (input). Coimmunoprecipitation of NSP1-3xHA indicated an interaction of NSP1 with itself.
(B) In a similar manner, N. benthamiana leaves were transformed with NSP2-3xHA or NSP2∷GFP alone, NSP2∷3xHA and NSP2-FLAG, or NSP2-GFP and NSP2∷FLAG. NSP2-FLAG was immunoprecipitated (elution) from total protein extracts (input) 3 d after infiltration. Coimmunoprecipitation of NSP2-GFP and NSP2-3xHA with NSP2-FLAG revealed homopolymerization of this protein. The input shown is from the NSP2-FLAG/NSP2-3xHA experiment. Similar inputs were observed with the NSP2-FLAG/NSP2-GFP experiment.
(C) Leaves of N. benthamiana were transformed with YFPN-NSP1 and YFPC-NSP1. Yellow fluorescence in the nuclei indicates homopolymerization of NSP1. Bar = 40 μm.
- Figure 4.
NSP1-NSP2 Heteropolymerization Is Relevant for Nodulation Signaling.
(A) and (B) M. truncatula nsp2-2 roots were transformed with NSP2 (A) and NSP2ΔLHRI (B) under control of the 35S promoter. Root systems were inoculated with S. meliloti and nodule formation monitored for up to 5 weeks. Transgenic roots were identified via fluorescence of GFP encoded on the binary vector. Transgenic Medicago roots harboring NSP2ΔLHRI did not develop nodules (B), while roots harboring NSP2 form wild-type-like nodules (A). The inset of (B) confirms the presence of (a) NSP2 and (b) NSP2ΔLHRI by PCR: the lower band of 522 bp represents the deleted form of NSP2 in the nsp2-2 mutant, while the upper represents either full-length NSP2 (984 bp) or NSP2ΔLHRI (780 bp). Bars = 5 mm.
(C) Quantification of β-galactosidase activity in S. cerevisiae revealed a threefold reduction in the heteropolymerization between NSP1 and NSP2 when a single amino acid change (A168V) is present in the LHRI domain of NSP2. One unit of β-galactosidase is defined as the amount that hydrolyzes 1 mmol o-nitrophenyl β-D-galactopyranoside per min. Each value is the mean of three technical and three biological replicates. Error bars represent se.
(D) M. truncatula nsp2-2 roots were transformed with NSP2A168V and NSP2 as control. The nodule number of transgenic roots that show GFP fluorescence was determined 35 d after inoculation with S. meliloti. Independent transformed roots examined were n = 75 (NSP2A168V) and n = 66 (NSP2).
(E) Nodules obtained from roots transformed with NSP2A168V and NSP2 as control were tested for their ability to fix nitrogen. The introduction of the amino acid change led to a severe decrease of nitrogen fixation as measured by rates of acetylene reduction (calculated as the amount of ethylene produced per hour and per milligram of nodule fresh weight). Each value is the mean of three biological replicates. Error bars indicate the se of the replicates.
- Figure 5.
NSP1 Associates with ENOD Promoters in Vitro.
(A) to (C) EMSA with NSP1 and NSP2 proteins using radiolabeled ENOD promoter probes. The promoter regions of ENOD11 (−1046 to +3) (A), NIN (−892 to −13) (B), and ERN1 (−862 to −29) (C) were used. The free ENOD promoter probe is present at the bottom of each image. The band shift, indicated with an arrow, revealed NSP1 association with the promoter of ENOD11 (A), NIN (B), and ERN1 (C) that is absent in the GST control and in NSP2. In (A), unlabeled ENOD11 fragment was used as competitor DNA. c10x and c50x, unlabeled competitor DNA in 10- and 50-fold excess, respectively.
(D) RT-PCR showing that the S. meliloti induction of NIN and ERN1 is absent in nsp1-1 and nsp2-2 mutants. Actin was amplified as a control. dpi, days post inoculation.
- Figure 6.
NSP1 Binds Specifically to an AATTT Motif Present in the ENOD11 Promoter.
(A) Identification of the NSP1 cis-element by random binding site selection (RBSS). NSP1 was found to bind to the indicated 15 oligonucleotides from a pool of 420 random-sequence oligonucleotides. All oligonucleotides bound to NSP1 contained the AATTT motif.
(B) The −411 to −257 region of the ENOD11 promoter contains two AATTT motifs (underlined). Regions around these motifs were synthesized and named NRE1 and NRE2.
(C) EMSA analysis showed that NSP1 binds to NRE1 and NRE2. Mutation of the AATTT motif to CCCCC in both NRE1 and NRE2 (indicated as NRE1-C5 and NRE2-C5) caused a dramatic reduction in NSP1 binding.
(D) A synthetic promoter was generated by fusing the minimal ENOD11 promoter lacking Nod factor inducibility to the NRE2 element and used to drive expression of GUS. This construct was transformed into plants and assessed for Nod factor inducibility, measured by a fluorimetric GUS assay. Nod factor inducibility was observed that was absent in nsp1-1 mutants and when using NRE2-C5. The asterisk indicates a significant difference to all other data points, measured with a t test at 95% confidence. NF, Nod factor. Values are means ± sd (n = 15 plants for each treatment).
- Figure 7.
NSP1 and NSP2 Associate with the ENOD11 Promoter in Vivo.
ChIP followed by PCR show in vivo association of both NSP1 and NSP2 with the ENOD11 promoter. NSP1 and NSP2 were purified from M. truncatula root extracts using their native antibodies; c-Myc antibodies were used as a negative control. Pretreatment with Nod factor enhanced the binding of NSP1 to the ENOD11 promoter and revealed an NSP2 association that we propose is via the interaction with NSP1. An equivalent analysis in nsp1-1 and nsp2-2 mutants revealed no association of NSP1 or NSP2 with the ENOD11 promoter.
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Author Profile
Sibylle Hirsch, Jiyoung Kim, and Alfonso Mu�oz
Sibylle Hirsch
Current Position:4 Year Rotation Student in the Department of Disease and Stress Biology at the John Innes Centre, Norwich, UK
Education: MSc in Biology (2004) at the Martin-Luther-University in Halle-Wittenberg, Germany
Non-scientific Interests: Ballroom and Latin American Dance, Pilates, Reading and Going out
When I was an undergraduate student at the Martin-Luther-University Halle-Wittenberg in Germany I was most interested by the lectures on Plant Physiology and Genetics and particularly loved to participate in the seminars held on Plant Biotechnology and Innovative Molecular Methods. I immediately decided to do my MSc, studying the genetic and molecular role of an effector protein from Pseudomonas syringae in the laboratory of Prof. Ulla Bonas in Halle. Intrigued by plant-microbe interactions and with the drive to broaden my knowledge by going abroad, I was very happy when I was offered the opportunity of an internship in Dr. Giles Oldroyd�s laboratory at the John Innes Centre in Norwich. I really enjoyed my time studying the legume-rhizobial symbiosis so much so that I started my PhD soon after, with a focus on Nod factor signal transduction in the plant. I am now specifically trying to understand the function of two GRAS domain proteins, NSP1 and NSP2, which are core components of the Nod factor signaling pathway. I was very excited to find that NSP1 and NSP2 interact physically in planta, and that this interaction is important for the formation of nodules and effective nitrogen fixation.Jiyoung Kim
Current Position: Ph.D. student
Education: M.S. in Molecular Biology, School of Life Sciences and Biotechnology, Korea University, Seoul, South Korea
Non-scientific Interests: Watching movies & playing badminton
I was born in Busan located on the Southeastern tip of the Korean Peninsula. I graduated with B.S. and M.S. in Plant Molecular Biology from Korea University. Many plant species acquire a significant amount of their nutritional needs through symbiotic interactions with micro-organisms. Bacterial nitrogen fixation provides a source of nitrogen to the plant. This interaction involves a molecular communication between the plant and the rhizobium. My work is focused on understanding how several putative transcription factors mediate this symbiotic interaction. Personally, it is a great pleasure to work with a scientist like Giles Oldroyd as a supervisor and I would like to thank the John Innes Centre and Giles Oldroyd for the opportunities and support that I've had in UK since 2006.
Alfonso Mu�oz
Current Position: Researcher, Department of Plant Molecular Genetics, National Centre for Biotechnology-CSIC in Madrid (Spain)
Education: PhD.: Department of Biochemistry and Molecular Biology. University of Cordoba, Spain (2003)
Non-scientific Interests: Reading, basketball, cycling, music and watching movies
After finishing my degree in Biological Sciences, I joined Dr. Manuel Pineda�s lab to do my PhD. on the biochemistry of ureide assimilation in legumes. The ureides allantoin and allantoate are the main compounds that transport recently fixed nitrogen in the nodules to the aerial part of some important legumes, such as soybean, French bean and cowpea. In this way, I became very interested on how this fixed nitrogen had been generated due to the establishment of the symbiosis between legumes and rhizobia. For that reason, I moved to Norwich to work on the first steps of the establishment of this symbiosis, first in Dr. Martin Parniske�s lab and later in Dr. Giles Oldroyd�s (corresponding author). Here, I developed several biochemical tools to study the biochemical relations and activities of the GRAS proteins NSP1 and NSP2 and showed their interactions by coimmunoprecipitation experiments. In collaboration with the other co-first authors, we showed the importance of the NSP1-NSP2 complex and its binding to the promoters of Nod factor-induced genes.
