Table 15.

Summary of key studies performed for assessing prospects for rice forming legume-like symbioses, and progress made in transfering nif^a genes to non-diazotrophic hosts including plants.

Highlights	References
Predisposition of rice for forming N2-fixing symbiosis with rhizobia
•Some rice cultivars exude compounds in root exudates that induce transcription of the nodb genes of Rhizobium species	Reddy et al. (2000); Rolfe et al. (2000)
•Bioengineering of rice plant to produce nod gene-inducing flavonoids in roots	Sreevidya et al. (2006)
•Expression of the legume symbiosis-related lectin (PSL) and lectin nucleotide phosphohydrolase (GS52/GsLNP) genes in rice supported improved intercellular infection/colonization in roots	Sreevidya et al. (2005)
•Evidence for the widespread occurrence of the homologs of early nodulin genes, and common symbiotic pathway genes of legumes in rice	Reddy et al., 1998a, Reddy et al., 1999; Kouchi et al. (1999);Goff et al. (2002); Yu et al. (2002); Gutjahr et al. (2008); Yano et al. (2008); Chen et al., 2007, Chen et al., 2008, Chen et al., 2009; Banba et al. (2008); Markmann and Parniske (2008)
•Demonstration of ability of rice roots to perceive nod factors (NF)	Reddy et al., 1998b; Liang et al. (2013); Altúzar-Molina et al. (2020)
•Expression of NF receptor proteins in rice confers root hairs the ability to respond to NFs in terms of exhibiting deformations
nifgene transfer to non-diazotrophic hosts including plants
•Transfer of nif gene cluster from N2-fixing Klebsiella pneumoniaein to Escherichia coli	Dixon and Postgate (1972)
•Transfer of iron-only (Anf) nitrogenase system composed of defined anf and nif genes from Azotobacter vinelandii into Escherichia coli	Yang et al. (2014)
•Transfer of Pseudomonas stutzeri nitrogen fixation island enables expression of active nitrogenase in Escherichia coli	Han et al. (2015);Zhang et al. (2015b)
•Expression of refactored Klebsiella oxytoca/ Klebsiella pneumonia nif gene cluster in Escherichi acoli	Temme et al. (2012); Wang et al. (2013); Smanski et al. (2014); Li et al. (2016)
•Transfer of a nif cluster from either Rhodobacter sphaeroides or Klebsiella oxytoca to generate free living N2-fixing Rhizobium sp IRBG74, and development of ammonium tolerant and oxygen tolerant N2-fixing Pseudomonas protegens Pf-5 by transferring nif cluster from Pseudomonas stutzeri and Azotobacter vinelandii	Ryu et al. (2020)
•Engineering N2 fixation activity in Synechocystis 6803 by transferring nif gene cluster from Cyanothece ATCC 51142 or Leptolyngbya boryana dg5	Liu et al. (2018); Tsujimoto et al. (2018)
•Transfer of the nif genes from Klebsiella pneumoniae to yeast	Zamir et al. (1981); Berman et al., 1985a, Berman et al., 1985b; Holland et al. (1987)
•Generation of active Fe protein by targeting A. vinelandii NifH and NifM to mitochondrial matrix, and by expressing NifH, NifM, NifS and NifU in the cytosol of yeast	Lopez-Torrejon et al., 2016
•Formation of nitrogenase NifDK tetramers in the mitochondria of •yeast by targeting Azotobacter vinelandii NifH, NifD, NifK, NifU, NifS, NifM, NifE, NifN, and NifB	Burén et al. (2017a)
•Active Methanocaldococcus infernus NifB could be produced in yeast mitochondria when co-targetted with A. vinelandii NifU, NifS, and FdxN	Burén et al., 2017b, Burén et al., 2019
•Production of FeMo (NifDK) tetramer and the active Fe protein in yeast by simultaneously transforming codon optimized nifH, nifD, nifK, nifB, nifE, nifN, nifV, nifX, hesA, groES, groEL of Pseudomonas polymyxa WLY78 and nifF, nifJ, nifS, nifU of Klebsiella oxytoca genes	Liu et al. (2019)
•Biosynthesis of cofactor-activatable iron-only nitrogenase (AnfH) in mitochondrial matrix of yeast	López-Torrejón et al. (2021)
•Identification of superior Hydrogenobacter thermophilus NifH protein variant to engineer N2 fixation in yeast and plants	Jiang et al. (2021)
•Development of NifD variant for its stable maintenance in mitochondrial matrix of eukaryotic cells (yeast/tobacco)	Allen et al. (2020); Xiang et al. (2020)
•Variations in solubilities of Nif proteins in tobacco mitochondrial environment have been identified (soluble components – NifF, M, N, S, U, W, X, Y and Z; insoluble components – NifB, E, H, J, K, Q and V). The limitations imposed by insolubility of some Nif proteins need to be overcome for successful assembly of nitrogenase in plant mitochondria	Okada et al. (2020)
•Targeting and expressed NifH protein together with NifM into chloroplasts of tobacco could generate functional NifH, although with low activity	Ivleva et al. (2016)
•Production of active nitrogenase Fe protein (NifH) by simultaneously targeting nuclear encoded Azotobacter vinelandii NifH, M, U and S components into chloroplasts of tobacco leaf cells	Eseverri et al. (2020)
•Demonstration of the feasibility of targeting and transient expression of the complete range of 16 biosynthetic and catalytic nitrogenase (Nif) proteins in tobacco leaves	Allen et al. (2017)

*In the case of nif gene transfer, the studies were ordered according to bacteria, cyanobacteria, yeast and plants.

**All results concerned with nif gene expression in plants are obtained with transient expression studies excepting in the investigation conducted by Ivleva et al. (2016), where they were achieved with the plants harboring stably transformed chloroplasts.

^anif = N₂ fixation;

^bnod= nodulation.