5.4.5 Genes for Protein Secretion
into Host Cells
The three symbiosis islands contain gene clusters
for secretion systems to deliver proteins into host
cells, although the cluster in MAFF303099
encodes a type III secretion system (T3SS),
whereas those in R7A and NZP2037 encode type
IV secretion systems (T4SS). These secretion
systems are not required for the nodulation of
L. japonicusbut do have negative and positive
effects on the nodulation of some other host
species (Hubber et al. 2004 ; Okazaki et al. 2010 ;
Sanchez et al. 2012 ).
MAFF303099 possesses a number ofttsgenes
orrhc(rhizobium-conserved) genes that encode
the secretion machinery as well asnop(nodula-
tion outer protein) genes that encode putative
secreted effector proteins. Therhcgenes together
with the regulatoryttsIgene are also conserved
in several other rhizobia includingRhizobiumsp.
NGR234 (Freiberg et al. 1997 ),Sinorhizobium
frediiUSDA257 (Krishnan et al. 2003 ),S. fredii
HH103 (Marie et al. 2001 ; de Lyra et al. 2006 ),
B. japonicumUSDA110 (Krause et al. 2002 ),
andB. elkaniiUSDA61 (Okazaki et al. 2009 ).
The repertoire of Nops differs depending on the
rhizobial species. Mutagenesis of mlr6361
enables nodulation onLotus halophiluswhich
wild-type MAFF303099 is unable to nodulate,
indicating that its product is a negative effector
(Okazaki et al. 2010 ).
In R7A and NZP2037, thevirB1–virB11and
virD4genes encode the secretion machinery of
T4SS (Hubber et al. 2004 ), whilevirAandvirG
encode a regulatory system that is itself under the
control of the NodD regulatory protein that acti-
vatesnodgene expression (Hubber et al. 2007 ).
R7A and NZP2037 share some of T4SS effector
proteins including Msi059 and Mln450 that are
negative effectors that prevent nodulation ofL.
leucocephala (Hubber et al. 2004 ). Notably,
Msi059 also shares strong similarity with the
T3SS effector Mlr6316 in MAFF303099 that
is also a negative effector for nodulation of
L. leucocephala(Hubber et al. 2004 ). This com-
monality and the presence ofvirgene remnants in
MAFF303099 (Sullivan et al. 2002 ) suggest that
the T3SS gene cluster in MAFF303099 replaced a
T4SS gene cluster in a more ancient symbiosis
island similar to that in R7A and NZP2037.5.5 Regulation of Symbiotic Genes5.5.1 Regulation of Genes Involved
in Nod Factor SynthesisIn all the threeM. lotisymbiosis islands, there are
two copies ofnodDwith syntenic positioning:
nodD1is monocistronic and possesses no obvi-
ous upstream element, whereasnodD2is situated
nearnodD1but in the opposite direction and may
be cotranscribed with the upstreamnolLgene that
is preceded by anod-box. NodD is a transcrip-
tional regulator that, when bound to a plant-
derivedflavonoid(s), binds tonod-boxes that are
usually located upstream of othernodgenes that
participate in the synthesis of LCOs. The con-
sensus sequence of thenod-box, 5′-YATCCAY-
NNYRYRGATGNNNNYNATCNAAACAAT-
CRATTTTACAATCY-3′, is conserved among
rhizobia (Schlaman et al. 1998 ); hence, it can be
possible to activatenodgenes using heterologous
nodDgenes and flavonoid molecules. In fact,
M. lotistrains harboringRhizobium leguminosa-
rum nodDgene synthesize LCO when naringe-
nin, the peaflavonoid that inducesnodgenes
inR. leguminosarumbv.viciae, is added (Lopez-
Lara et al. 1995 ; Niwa et al. 2001 ). In R7A,
disruption of bothnodD1andnodD2results in no
nodulation (Nod−phenotype), while disruption of
nodD1 gives delayed nodulation and that of
nodD2has no apparent phenotype withLotus
species (Rodpothong et al. 2009 ). However,
nodD1 disruption alone abolishes nodulation
capacity withL. leucocephala(Rodpothong et al.
2009 ). These observations indicate thatnodD1
and nodD2 are functionally exchangeable,
althoughnodD1is more important thannodD2.52 K. Saeki and C.W. Ronson