non-legumes, followed by recruitment of NLP2,
to enhance LgHb expression in legume nodules.
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ACKNOWLEDGMENTS
We thank X. Fang for providing purified NIN protein andnlp1mutant
seeds, J. Feng for providing seeds ofnlp2-1, D. Guan and F. Robson
for their contributions early in the project, and G. Oldroyd (University of
Cambridge) and the ENSA program for providing plasmid backbones
for Golden Gate cloning andM. truncatularoot transformation.
Funding:Supported by the CAS Project for Young Scientists in Basic
Research (YSBR-011), National Science Foundation China
(3217020272), Ministry of Science and Technology (2019FA0904703),
Strategic Priority Research Program of the Chinese Academy
of Sciences (XDB27040209), National Key R&D Program of
China (2016YFA0500500), and Chinese Academy of Sciences
(153D31KYSB20160074). Also supported by Biotechnology
and Biological Sciences Research Council grants BB/L010305/
1 (David Phillips Fellowship), BB/J004553/1 (J.M.), and BB/N003608/1 (C.S.C.); China Postdoctoral Science Foundation grant
2018M642099 (S.J.); ANR grants EPISYM (ANR-15-CE20-0002)
and Laboratoire d’Excellence (LABEX) TULIP (ANR-10-LABX-41)
(P.G., M.F.J., and Y.P.); and NSF grants DBI-0703285 and IOS-1127155
(K.M. and J.W.).Author contributions:The experiments were
conducted by S.J., M.-F.J., J.-P.G., Y.P., J.W., F.L., C.S.C., Q.L., Y.T.R.,
and M.Z.; K.M. and J.W. generated theTnt1insertion mutants;
experiments were designed by S.J., M.-F.J., J.-P.G., P.X., P.G., and
J.D.M.; and P.X., P.S.P., E.W., P.G., and J.D.M. wrote the manuscript.
Competing interests:The authors declare no competing interests.
Data and materials availability:All data are available in the
main text or the supplementary materials.SUPPLEMENTARY MATERIALS
science.org/doi/10.1126/science.abg5945
Materials and Methods
Figs. S1 to S14
Tables S1 to S5
References ( 22 Ð 34 )
Data S1
MDAR Reproducibility Checklist
15 January 2021; accepted 16 September 2021
10.1126/science.abg5945628 29 OCTOBER 2021¥VOL 374 ISSUE 6567 science.orgSCIENCE
Fig. 3. NLP2 directly activates
LgHbexpression via NRE-like
promoter elements.(A)“dNRE”
motif detected in promoters of
M. truncatula nlp2-1down-regulated
genes compared with NREs detected
inNiRsacrossangiospermplants
(this study). The gapped semi-
palindrome is indicated by arrows.
Overlapping NREs are indicated by
brackets. Matches and mismatches of
the NRE consensus to the dNRE are
indicated by asterisks and dashes,
respectively. (B) The position of the
dNRE sequence in promoters of
M. truncatula LgHbs (NRE1 in orange,
NRE2 in white). The bold line indicates
the 5′-untranslated region. TSS,
transcription start site; ATG, start
codon. (C)EMSAtotestfor
interaction between a truncated NLP2
protein containing the RWP-RK
domain with an N-terminal His-tag
and the CY5-labeledLgHb7promoter
fragment containing the dNRE.
M:LgHb7promoter with a mutated
dNRE. (D) EMSA to test for interac-
tion between a truncated NIN
protein containing the RWP-RK
domain with an N-terminal His tag
and the CY5-labeledLgHb7
promoter fragment containing the
dNRE. M:LgHb7promoter with
deleted dNRE. (E) Relative expres-
sion (qRT-PCR) ofLgHb1 and LgHb2
in nodules ofM. truncatulaplants
transformed with a CRISPR construct
with two gRNAs targeting the dNRE
ofLgHb1. gRNA1 also matches the dNRE sequence in theLgHb2promoter, but
there is no corresponding PAM site. The vector was introduced by hairy-root
transformation; gene expression was evaluated at 21 dpi withS. melilotiRm2011.
EFwas used as a reference gene. (F) Trans-activation assay for NLP2 and the
LgHb7promoter drivingGUSinAgrobacterium tumefaciens–co-infiltratedNicotiana
benthamianaleaves. EV1, empty vector no-promoter control; EV2, empty vector
no-NLP2control;pLgHb7,LgHb7promoter fragment (–342 to–1 bp) containing
the dNRE;LgHb7m,LgHb7promoter fragment with the dNRE deleted;NINand
NLP2, p35S promoter driving full-lengthNLP2orNIN. In (E) and (F), data are
means ± SD and are compared by Student’sttest,a= 0.05. In (E),n= 3; in (F),
each replicate consisted of six leaves. For NLP2 experiments,n= 3 or 4; for
NIN experiments,n= 6 to 8. In (E) and (F), *P< 0.05; ns, not significant.RESEARCH | REPORTS