Bacillus velezensis strain B26 modulates the inflorescence and root architecture of Brachypodium distachyon via hormone homeostasis | Scientific Reports –


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AbstractPlant growth-promoting rhizobacteria (PGPR) influence plant health. However, the genotypic variations in host organisms affect their response to PGPR. To understand the genotypic effect, we screened four diverse B. distachyon genotypes at varying growth stages for their ability to be colonized by B. velezensis strain B26. We reasoned that B26 may have an impact on the phenological growth stages of B. distachyon genotypes. Phenotypic data suggested the role of B26 in increasing the number of awns and root weight in wild type genotypes and overexpressing transgenic lines. Thus, we characterized the expression patterns of flowering pathway genes in inoculated plants and found that strain B26 modulates the transcript abundance of flowering genes. An increased root volume of inoculated plants was estimated by CT-scanning which suggests the role of B26 in altering the root architecture. B26 also modulated plant hormone homeostasis. A differential response was observed in the transcript abundance of auxin and gibberellins biosynthesis genes in inoculated roots. Our results reveal that B. distachyon plant genotype is an essential determinant of whether a PGPR provides benefit or harm to the host and shed new insight into the involvement of B. velezensis in the expression of flowering genes.

IntroductionBacillus species are one type of rhizobacteria that can boost plant growth through the induction of antibiosis, facilitating nutrient availability through the synthesis of phytohormones, and competitive omission1. Such interactions help in endurance and adaptation of both host and PGPR in any stress environment2. We previously demonstrated that Bacillus velezensis strain B26, is a growth-promoting bacterium of timothy grass and the model plant Brachypodium distachyon, which enhanced the growth and accelerated flowering time through the production of hormones, volatiles and various antimicrobial compounds3,4. We also showed that strain B26 improves the growth of these grasses under extended drought conditions by modulating the expression of drought-responsive genes in B. distachyon, and also by the modification of osmolytes in roots and shoots of timothy grass3. Successful colonization of B. distachyon roots by strain B26 is based on the composition of roots exudates (the type of organic acid and their biosynthetic genes), chemotaxis and the induction of biofilm and their encoding genes5.It is well established that plant genotype can impact the degree of plant growth-promotion of some PGPR6. The effects of inoculation of 20 rice cultivars of genetically distinct groups with Azospirillum sp. provided varied results in terms of the number of tillers7. Also, different accessions of Arabidopsis displayed different microbial communities, indicating that plant host genetic factors shape the associated microbiota6,8. The genotypes of the model grass B. distachyon has an important role in defining the plant host responses to PGPR9. However, it is unclear whether the host’s genotypic variations affect the microbiome in such a way that leads to adaptive consequences to the host. The study of Do Amaral et al.9 and others only described the short-term growth responses on plants10.B. distachyon is closely related to cultivated monocotyledons such as rice, wheat, and maize, and is a model plant to study plant–microbe interactions and stress tolerance4,11,12. Due to ease in genetic transformation, B.distachyon is ideal for generating transgenic lines13. Various transgenic lines have been generated in the background of B.distachyon accession line Bd21-3 with loss and gain of function of a target gene14,15. Moreover, B. distachyon accessions exhibit variation in various phenotypic traits16.The reproductive success of many plants hinges on flowering17. Flowering responds to environmental cues such as long exposure to cold temperatures (i.e., vernalization) and photoperiods (i.e., variation in day length). The regulation of the flowering process in B. distachyon is controlled by several key genes, which include VERNALIZATION 1 (VRN1), VRN2 and FLOWERING LOCUS FT1 (FT1)17,18,19.The expression of these genes is affected by temperature and photoperiods20. It was demonstrated that the over-expression of FT1 accelerates flowering in B. distachyon and wheat17,21. However, the flowering pathways are not limited to the shoot apical meristem where flowers are originated, but it depends on shoot–root communication22,23. For example, the majority of flowering genes in Arabidopsis and Cassava are variably expressed when plants are exposed to photoperiod that induces flowering22,23.These studies provide a new understanding on the involvement of the root in the flowering process. Signalling molecules from roots including phytohormones modulate shoot growth and root architecture24. Additionally, the plant growth stimulation by beneficial rhizobacteria has been associated with the biosynthesis of plant growth regulators produced by rhizobacteria including auxins, gibberellins, cytokinins and ABA25. These microbial signals alter the plant hormone levels. Previously, we reported on the beneficial traits mediated by phytohormones produced by B. velezensis strain B264 causing increased fitness of plant resulting in 121% more spikelets in inoculated B. distachyon than the respective control3. Despite significant advances in plant-rhizobacteria interactions, regulation of plant flowering genes in response to rhizobacteria is scarce26.Here, we aim to (i) study the potential use of B. distachyon genotypes for studies of PGPR-grass interactions throughout the whole growth cycle of the genotypes. (ii) characterize the responses of expression patterns of selected flowering genes to B. velezensis inoculation in Brachypodium wild accessions and (iii) understand whether strain B26 could alter the expression of Brachypodium transgenic lines overexpressing flowering genes relative to the colonized wild type (iv) understand whether growth promotion by strain B26 is differentially associated with phytohormone homoeostasis and transcript abundance. We screened four diverse genotypes of Brachypodium for their ability to be colonized by B.velezensis. We reasoned that B. velezensis may have an impact on the inflorescence and root architecture of B. distachyon genotypes.ResultsBacterial inoculation elicited varied growth response of B. distachyon accessionsA differential response was observed in Bd21, Bd21-3, Bd18-1 and Bd30-1 in response to B26 colonization (Fig. 1a). At 14 days post inoculation (dpi), a significant increase of 150% in the number of awns and 250% increase in the shoot weight of inoculated accession Bd21 compared to non-inoculated control was observed (Fig. 1b, Table S2). The plant height and number of leaves of inoculated Bd18-1 increased by 34% and 78%, respectively compared to the control. At 28 dpi, Bd21-3 showed a significant increase in all growth parameters compared to the control (Fig. 1c). While Bd 30-1 at 28dpi, did not show a significant response to B26 inoculation as indicated by the growth parameters (Fig. 1c). However, there was no difference in flowering time of inoculated and non-inoculated plants. Control and inoculated accessions flowered at the same time but an increase in the number of awns was observed.Figure 1(a) Brachypodium accession lines displaying growth response at 28 days post-inoculation (dpi). The left panel of (a) shows accession lines (Bd21-3, Bd21, Bd18-1 and Bd30-1) inoculated with Bacillus velezensis strain B26. The right panel shows control accession lines (b) Growth response parameters (Plant height, No. of leaves, No. of tillers, No, of awns, Root weight and shoot weight) of wild type B.distachyon genotypes in response to B26 inoculation at 14 days post-inoculation (dpi) (c) at 28 dpi. Bars represent the mean of five biological replicates. t-test was used to determine statistical differences between inoculated and non-inoculated plants. * indicates significance according to Independent Student t-test (p