Adaptation and regulation of the alternative lifestyle of insect pathogenic Photorhabdus luminescens in the soil and its potential as biocontrol agent
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Abstract
Soil living and plant beneficial bacteria rose in importance as biocontrol agents in sustainable agriculture as many pests and diseases harshly reduce crop yields. Photorhabdus luminescens is a Gram-negative bacterium living in symbiosis with entomopathogenic nematodes (EPNs) and is highly pathogenic towards a wide range of insect larvae. The EPNs-Photorhabdus complex is already employed in agriculture as biocontrol agent. P. luminescens exists in two phenotypically different forms, the primary (1°) and the secondary (2°) cell variants, however only the 1° cells live in mutualistic symbiosis with EPNs. Once the nematodes invade insects and release P. luminescens into the hemocoel, the bacteria effectively kill the larvae. During the infective lifecycle up to 50% of 1° cells switch to the 2° phenotype. Since 2° cells cannot reassociate with EPNs they are left in soil when the insect cadaver is depleted. Both cell variants are believed to share identical genomes, but they differ in many phenotypic traits, which is referred to as phenotypic heterogeneity. However, the fate of 2° cells in the soil and therefore the biological reason for phenotypic heterogeneity is unclear. Moreover, the genetical identity of both cell variants has not been confirmed yet. For that purpose, this work focuses on the biological role of 2° cells in the rhizosphere.
First, to understand the regulation processes that are involved in phenotypic switching and to obtain a first idea for the fate of 2° cells a comparative transcriptome analysis of P. luminescens DJC 1° cells and 2° cells was performed. First of all, it could be proved that the different 1° and 2° specific phenotypes are regulated at transcriptional level. In fact, the respective genes coding for 1°-specific features like e.g., pigmentation, bioluminescence, clumping factors were downregulated in 2° cells. Furthermore, differently expressed genes (DEGs) coding for different LuxR solos were identified, indicating that a yet unknown circuit of cell-cell communication could exist in 2° cells. For example, the major regulator of quorum sensing (QS) in 1° cells, pluR, was downregulated in 2° cells, whereas genes encoding PAS4-LuxR-solos PluDJC_10415-PluDJC_10460 and two LuxR-solos with an undefined signal binding domain PluDJC_09555 and PluDJC_21150 were upregulated in 2° cells. This also points out a putative regulatory role of QS in P. luminescens phenotypic heterogeneity.
Furthermore, DEGs involved in stress-response such as starvation related genes were upregulated, while genes involved in metabolism were differently modulated in 2° cells
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indicating an adaptation to the nutrient-limited availability in the soil. Moreover, increased swimming and twitching motility as well as chemotaxis, which are essential for rhizosphere colonization, were observed for 2° cells. This supports the hypothesis of an alternative lifecycle of P. luminescens in the rhizosphere. Remarkably, 2° cells chemotactically responded to plant root exudates (PRE), showed increased biofilm formation, and specifically interacted with plant roots. Additionally, plant growth promoting ability of this cell variant could be determined. To further understand the adaptability of 2° cells to plant roots, a comparative transcriptome analysis was performed comparing 2° cells supplemented with and without PRE. Here, DEGs involved in e.g., biofilm formation, motility, or chitin degradation were identified to be upregulated in the presence of PRE. Two of the most upregulated genes were those encoding a putative chitin binding protein and a putative chitinase (Chi2A) suggesting the chitin degrading and therefore fungicidal activity of 2° cells in the soil. Indeed, 2° cells specifically inhibited growth of phytopathogenic Fusarium graminearum after physical contact. This ability was impaired in P. luminescens 2°
Δchi2A and Δcbp deletion mutants. Furthermore, in planta assays using tomato plants infected with F. graminearum proved that 2° cells could protect the plant from infection and therefore promoted plant growth, which was not the case using P. luminescens 1° wildtype and the 2° Δchi2A and Δcbp deletion mutants. Moreover, effective chitin degradation was verified using purified Chi2A enzyme. This indicates a role of 2° cells in protecting plants from phytopathogens upon root colonization.
Moreover, a SdiA-like LuxR solo was identified as an essential player in interkingdom signaling (IKS) communication between plants and the bacteria. SdiA could play a role in the first steps of root colonization as a decreased motility and increased biofilm formation of P. luminescens 2° ΔsidA was observed compared to wildtype 2° cells. A plant-derived signaling molecule is assumed to be sensed by SdiA, which could lead to expression of genes important for the 2° cells-plant interaction. Furthermore, putative binding of long and short chain N-acyl homoserine lactones (AHLs) to SdiA were suggested, as different folding conformations occurred upon binding. Using surface plasmon resonance spectroscopy a direct and high affine interaction of purified SdiA to its own promoter as well as to the promoter of the gene adjacent to sdiA, aidA, could be demonstrated. This indicated a bidirectional transcriptional regulation of the intergenic aidA-sdiA promoter region. Furthermore, a putative role of AidA in microbe-host interaction and an accurate self-regulatory mechanism of SdiA could be assigned.
Summary
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Lastly, to verify phenotypic heterogeneity in P. luminescens subs. laumondii DJC strain high-throughput sequencing data of the respective genomes were analyzed. With that the genetic similarity of both cell variants should be confirmed to exclude genotypic heterogeneity. Indeed, it could be confirmed that P. luminescens DJC 1° and 2° are genetical identical, and that large genome rearrangements are not involved in the switch from the 1° to the 2° phenotype.
In conclusion, the presented thesis gives direct evidence for an alternative lifestyle of P. luminescens 2° in the rhizosphere for the first time. The bacteria show a specific adaptation to plant roots protecting them from phytopathogenic fungi. Besides the biotechnological use of P. luminescens 1° cells as bioinsecticides, 2° cells could be used as plant growth promoting organism and as biopesticide for plant protection in the future.