Improving legume nodulation and Cu rhizostabilization using a genetically modified rhizobia

The rhizobia–legume interaction has been proposed as an interesting and appropriate tool for rhizostabilization of soils contaminated with heavy metals. One of the main requirements to use this symbiosis is the availability of tolerant and symbiotically effective rhizobia. The aim of this work was to improve the symbiotic properties of the arsenic-resistant wild-type strain <i>Ensifer medicae</i> MA11 in Cu-contaminated substrates. The <i>copAB</i> genes from a Cu-resistant <i>Pseudomonas fluorescens</i> strain were expressed in <i>E. medicae</i> MA11 under the control of the <i>nifH</i> promoter. The resulting strain <i>E. medicae</i> MA11-<i>copAB</i> was able to alleviate the toxic effect of Cu in <i>Medicago truncatula</i>. At 300 µM Cu, root and shoot dry matter production, nitrogen content, number of nodules and photosynthetic rate were significantly reduced in plants inoculated with the wild-type strain. However, these parameters were not altered in plants inoculated with the genetically modified strain. Moreover, nodules elicited by this strain were able to accumulate twofold the Cu measured in nodules formed by the wild-type strain. In addition, the engineered <i>E. medicae</i> strain increased Cu accumulation in roots and decreased the content in shoots. Thus, <i>E. medicae</i> MA11-<i>copAB</i> increased the capacity of <i>M. truncatula</i> to rhizostabilize Cu, decreasing the translocation factor and avoiding metal entry into the food chain. The plasmid containing the <i>nifH</i> promoter-<i>copAB</i> construct could be a useful biotool for Cu rhizostabilization using legumes, since it can be transferred to different rhizobia microsymbionts of authoctonous legumes growing in Cu-contaminated soils.