Sinorhizobium fredii HH103 nolR and nodD2 mutants gain capacity for infection thread invasion of Lotus japonicus Gifu and Lotus burttii

Abstract

Summary Sinorhizobium fredii HH103 RifR, a broad‐host‐range rhizobial strain, forms ineffective nodules with Lotus japonicus but induces nitrogen‐fixing nodules in Lotus burttii roots that are infected by intercellular entry. Here we show that HH103 RifR nolR or nodD2 mutants gain the ability to induce infection thread formation and to form nitrogen‐fixing nodules in L. japonicus Gifu. Microscopy studies showed that the mode of infection of L. burttii roots by the nodD2 and nolR mutants switched from intercellular entry to infection threads (ITs). In the presence of the isoflavone genistein, both mutants overproduced Nod‐factors. Transcriptomic analyses showed that, in the presence of Lotus japonicus Gifu root exudates, genes related to Nod factors production were overexpressed in both mutants in comparison to HH103 RifR. Complementation of the nodD2 and nolR mutants provoked a decrease in Nod‐factor production, the incapacity to form nitrogen‐fixing nodules with L. japonicus Gifu and restored the intercellular way of infection in L. burttii. Thus, the capacity of S. fredii HH103 RifR nodD2 and nolR mutants to infect L. burttii and L. japonicus Gifu by ITs and fix nitrogen L. japonicus Gifu might be correlated with Nod‐factor overproduction, although other bacterial symbiotic signals could also be involved.

Sinorhizobium fredii HH103 syrM inactivation affects the expression of a large number of genes, impairs nodulation with soybean and extends the host‐range to Lotus japonicus

“…In addition to nodD2 , another important regulatory gene is affected: nolR (= SFHH103_02239), a global regulator that participates in the HH103 nod regulon (Vinardell et al ., ; Acosta‐Jurado et al ., ), is repressed (fold‐change = −3.2) in the absence of SyrM and the presence of genistein.…”

“…In order to study whether the presence of the wild‐type copy of nolR or syrM gene could affect its own expression, we have also checked the expression of the nolR :: lacZ and syrM :: lacZ fusions in the mutant strains complemented with the wild‐type copy of the gene in cis . As previously shown for the complemented version of HH103 nolR :: lacZ ‐Gm R (Acosta‐Jurado et al ., ), introduction of the wild‐type version of syrM into HH103 syrM :: lacZ ‐Gm R restored the interactions with Lotus japonicus to those of the wild‐type strain (the absence of nitrogen‐fixing nodules). However, with L. burttii the complemented HH103 syrM :: lacZ ‐Gm R mutant exhibited a mixed phenotype: microcolonies on curled root hairs (as the wild‐type), but also some infection threads (as the mutant).…”

“…Sinorhizobium fredii HH103 invades L. burttii roots by intercellular infection but it is not able to invade L. japonicus Gifu roots (Acosta‐Jurado et al ., ). In both plants, at 21 dpi, S. fredii HH103 carrying pFAJDsRed was located forming microcolonies on curled root hairs (Fig.…”

“…In Lotus burttii , the wild‐type strain, instead, was found in intercellular spaces (Fig. , panel B5), as it was described previously (Acosta‐Jurado et al ., ).…”

“…Since one of the syrM mutants was constructed by insertion of lacZ ‐Gm R cassette, we could also analyse syrM expression during the infection process and inside developed nodules in the two species of Lotus tested as well as in soybean by checking β‐galactosidase activity in situ (Madsen et al ., ). A HH103 nolR :: lacZ ‐Gm R mutant, also able to induce formation of ITs in L. burttii and L. japonicus Gifu (Acosta‐Jurado et al ., ), was included in these experiments. At 21 dpi, both syrM and nolR expression was clearly detected in bacteria located inside ITs in the three legumes analysed (Supporting Information Fig.…”