of NIN in both species, leads us to conclude
that NIN is a target for the DNF1 complex in
both species and that NCR processing alone
cannot explain thednf1mutant phenotypes.
NINcontrols all aspects of symbiotic nodu-
lation: initiation of nodule organogenesis, in-
tracellular rhizobial infection, regulation of
nodule number ( 6 ), and now also the transi-
tion to nitrogen fixation. We show that one
mechanism allowing this single protein to con-
trol so many different functions is proteolytic
processing by the DNF1 SPC. The specific ex-
pression ofDNF1in the infection zone of the
nodule ( 12 ) likely allows transition of cells into
those that can support nitrogen fixation and
sustain bacterial infection. WhileNINis essen-
tial for early and late nodule-associated gene
expression,DNF1is essential only for the late
nodule-associated gene expression. Thus, pro-
cessing of NIN is only relevant for functions
late in nodule development, not for activation
of earlier genes such asCRE1andNPL. Among
the genes controlled byNINat late stages of
nodulation are leghemoglobins that buffer oxy-
gen ( 24 ), NCR peptides that drive bacteroid dif-
ferentiation ( 15 ), and thioredoxins that control
theredoxstateofthenodule( 25 ). NIN, as pro-
cessed by the DNF1 complex, is both necessary
and sufficient for the expression of these genes
and the transition to the nitrogen-fixing state.
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ACKNOWLEDGMENTS
We thank J. Murray, R. Dickstein, J. Stougaard, and J. Fournier
for providing strains and seeds and E. Barclay and K. Findlay for
support with microscopy.Funding:This work was supported by
the Bill and Melinda Gates Foundation and the UK Foreign,
Commonwealth and Development Office (OPP1028264) through
Engineering the Nitrogen Symbiosis for Africa (ENSA) project;
the Biotechnology and Biological Sciences Research Council
(BB/K003712/1); and the Gatsby Foundation (GAT3395/GLH).
Author contributions:J.F. and G.E.D.O. designed the
experiments, analyzed the data, and wrote the manuscript. J.F.
performed most of the experiments. K.S. performedNIN
overexpression. T.L. analyzed RNA sequencing (RNA-seq) data.
Competing interests:The authors declare no competing interests.
Data and materials availability:All data are available in the
main text or the supplementary materials. The RNA-seq data
generated in this study have been deposited at the National Center
for Biotechnology Information Gene Expression Omnibus under
accession number GSE178119.SUPPLEMENTARY MATERIALS
science.org/doi/10.1126/science.abg2804
Materials and Methods
Figs. S1 to S19
Tables S1 to S6
References ( 26 Ð 59 )
MDAR Reproducibility Checklist11 January 2021; accepted 27 August 2021
10.1126/science.abg2804632 29 OCTOBER 2021¥VOL 374 ISSUE 6567 science.orgSCIENCE
Fig. 4. DNF1-dependent NIN processing is conserved inL. japonicus.(A) Wild-type (WT) andLjdnf1
nodules 14 days afterM. lotiinoculation. (B) Bacterial NifH in the nodules of WT,Ljnin-2, andLjdnf1.
(C) TEM images showing symbiosomes in nodules of WT andLjdnf1. Arrows indicate bacterial membranes.
(D) LjNIN proteins in the nuclear extracts of WT andLjnin-2at the indicated time points after inoculation of
M. loti.(E) LjNIN in the membrane fractions (M) and nuclear fractions (N) of WT,Ljnin-2, andLjdnf1. Red
asterisks in (D) and (E) indicate NIN-specific products.
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