Abstract
ABSTRACTThe interior of plants contains microorganisms (referred to as endophytes) that are distinct from those present at the root surface or in the surrounding soil. Herbaspirillum seropedicae strain SmR1, belonging to the betaproteobacteria, is an endophyte that colonizes crops, including rice, maize, sugarcane, and sorghum. Different approaches have revealed genes and pathways regulated during the interactions of H. seropedicae with its plant hosts. However, functional genomic analysis of transposon (Tn) mutants has been hampered by the lack of genetic tools. Here we successfully employed a combination of in vivo high-density mariner Tn mutagenesis and targeted Tn insertion site sequencing (Tn-seq) in H. seropedicae SmR1. The analysis of multiple gene-saturating Tn libraries revealed that 395 genes are essential for the growth of H. seropedicae SmR1 in tryptone-yeast extract medium. A comparative analysis with the Database of Essential Genes (DEG) showed that 25 genes are uniquely essential in H. seropedicae SmR1. The Tn mutagenesis protocol developed and the gene-saturating Tn libraries generated will facilitate elucidation of the genetic mechanisms of the H. seropedicae endophytic lifestyle.IMPORTANCE A focal point in the study of endophytes is the development of effective biofertilizers that could help to reduce the input of agrochemicals in croplands. Besides the ability to promote plant growth, a good biofertilizer should be successful in colonizing its host and competing against the native microbiota. By using a systematic Tn-based gene-inactivation strategy and massively parallel sequencing of Tn insertion sites (Tn-seq), it is possible to study the fitness of thousands of Tn mutants in a single experiment. We have applied the combination of these techniques to the plant-growth-promoting endophyte Herbaspirillum seropedicae SmR1. The Tn mutant libraries generated will enable studies into the genetic mechanisms of H. seropedicae-plant interactions. The approach that we have taken is applicable to other plant-interacting bacteria.
Highlights
IntroductionThe interior of plants contains microorganisms (referred to as endophytes) that are distinct from those present at the root surface or in the surrounding soil
The interior of plants contains microorganisms that are distinct from those present at the root surface or in the surrounding soil
Several characteristics make H. seropedicae a suitable model endophyte [6], i.e., (i) it provides fixed nitrogen for important agroeconomic cultivars, (ii) it is genetically tractable, (iii) it has mechanisms of plant growth promotion other than nitrogen fixation, (iv) it has a wide range of plant hosts, (v) culturable bacteria are not isolated from soil and are isolated only from inside plants [7, 8], and (vi) there are publicly available genome sequences [8]
Summary
The interior of plants contains microorganisms (referred to as endophytes) that are distinct from those present at the root surface or in the surrounding soil. Different approaches have revealed genes and pathways regulated during the interactions of H. seropedicae with its plant hosts. The Tn mutant libraries generated will enable studies into the genetic mechanisms of H. seropedicae-plant interactions. Several characteristics make H. seropedicae a suitable model endophyte [6], i.e., (i) it provides fixed nitrogen for important agroeconomic cultivars, (ii) it is genetically tractable, (iii) it has mechanisms of plant growth promotion other than nitrogen fixation, (iv) it has a wide range of plant hosts, (v) culturable bacteria are not isolated from soil and are isolated only from inside plants [7, 8], and (vi) there are publicly available genome sequences [8]. Transcriptomic and proteomic approaches have identified genes and pathways that are regulated during the interactions of H. seropedicae with different plant hosts [12,13,14]. Comparative genomics and metagenomics studies have shown that certain functions, e.g., nutrient transport systems, type
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