Factors other than root secreted malic acid that contributes toward Bacillus subtilis FB17 colonization on Arabidopsis roots

Abstract

The plant growth promoting rhizobacterium (PGPR) Bacillus subtilis FB17 (hereafter FB17) induces resistance against broad pathogen including Pseudomonas syringae pv tomato (PstDC3000). The extent of plant protection by FB17 depends on establishment of root colonization followed by biofilm formation. The general convention dictates that beneficial rhizobacterium may suppress the root innate immune system to establish a robust colonization. However, it is still not well understood which genetic targets FB17 affects in plants to facilitate a symbiotic association. Our recent study, involving whole transcriptome analysis of Arabidopsis thaliana roots treated with FB17 post 24 h of treatment showed totally 279 genes that were significantly up- or/ downregulated. Further, we found that the mutants for upregulated and downregulated genes post-FB17 colonization showed a differential phenotype for FB17 root colonization. Interestingly, plants mutated in the FB17-responsive genes showed increased Aluminum activated malate transporter (ALMT1) expression under foliar pathogen PstDC3000, infections, indicating the independent functionality of ALMT1 for bacterial recruitment. Taken together this, present study suggests that the establishment of interaction between the plant host and PGPR is a complex phenomenon which is regulated by multiple genetic components.

The portion of the soil surrounding plant root, the rhizosphere retains dynamic communities of plant pathogenic and beneficial microorganisms. Plant growth-promoting rhizobacteria (PGPR) is described as bacteria that thrive in the rhizosphere, colonize the roots, and imparts various beneficial effect on plant growth.¹ The mechanisms of plant growth promotions by PGPR was proposed by several studies including, simultaneous activation of induced systemic responses (ISR) and systemic acquired responses (SAR), against pathogenic bacteria,^2-4 production of plant growth hormones,⁵ bacterial volatiles,⁶ and increased availability of soil nutrients to plants. The mechanism by which PGPR protects plants includes efficient root colonization and subsequent biofilm formation.^7-10 Of late, research has begun to elucidate the genetic pathways and molecular components that control root biofilm formation by B. subtilis. Plants use an array of metabolites to attract microorganisms that are considered beneficial. The accepted mechanism is that root exudates play an important role to initiate a rhizosphere dialog between plant and microbes.^7,11,12 Arguably, the root secreted metabolites do not only serve as carbon source for soil microorganisms, but also act as signal to attract or repel microbes.¹³ The organic acids also facilitate the chemotactic response in various beneficial rhizobacteria.¹⁴ Recent studies revealed that pathogenic infection or foliar treatment of MAMPS such as flg22 in shoot of A. thaliana specifically elucidate L-malic acid secretion in roots that specifically attract B. subtilis FB17.^8,15,16 Similarly, citric acid and malic acid in cucumber¹⁷ and watermelon;¹⁸ fumaric acid in banana root exudates help recruit rhizobacteria.¹⁸

On the other hand, PGPR Bacillus cereus AR156 is reported to suppress defense-related gene expression in Arabidopsis roots that may contribute to the stable root colonization.⁹ The other study also shows that Pseudomonas fluorescens WCS417r and FB17 suppresses MAMP-elicited defense responses in A. thaliana roots, such as deposition of callose in the root elongation zone elicited by the flagellar peptide flagellin (flg22).^15,19 These studies suggest that beneficial microorganisms actively block root innate immune responses in A. thaliana and establish a compatible interaction with their host, which may be important for root colonization. In this study, we performed whole root transcriptome analysis using the model plant A. thaliana to gain an insight into molecular changes in the host root induced by the PGPR FB17. Understanding the mechanism involved in plant root–beneficial microbe interaction will facilitate practical application of PGPR in agricultural practice.

Impact of Bacillus subtitis FB17 on plant root transcriptome

To determine changes in A. thaliana root transcriptome triggered by FB17 colonization, microarray analyses were performed post 24h of bacterial treatment. We identified 168 and 129 genes that were up and downregulated post FB17 root colonization. The up- and downregulated genes were distributed into 18 functional categories. To further verify, semi-quantitative RT-PCR assays were performed for 11 upregulated and 9 downregulated genes. Our results showed downregulation of 4 defense-related genes including modification of cell wall related genes that were critically modified during FB17 colonization. Interestingly, the above observation shows that PGPR binding to the roots results in morphological and biochemical changes at the cell wall level. This conclusion was further reinforced by experiment with evidence of differential FB17 colonization on the roots of mutants of upregulated genes and downregulated genes compared with FB17 colonization on Col-0 (Fig. 1). The results showed significantly reduced FB17 colonization on the mutants of upregulated genes at3g14460 (Disease resistance protein, NBS-LRR class), at3g20190 (Leucine-rich repeat transmembrane protein kinase), and at1g21240 (Wall-associated kinase, putative). Similarly, 3 mutants of downregulated genes at3g27980 (Pectinesterase family protein), at4g19690 (Iron-responsive transporter (IRT1), and at5g56320 (Expansin, EXP14) exhibited increased level of FB17 colonization compared with Col-0 wild type FB17-treated roots. Our results show that both cell wall modification and plant defense genes are primary targets of FB17. In addition, the modulation of both cell wall modification and plant defense genes bring changes in how FB17 colonizes the plant root surface. Therefore, further experimentation and detailed genetic characterization work must be done to identify and differentiate host specific and non-specific signals that induce the root colonization and biofilm formation on roots by FB17.

Figure 1. Bacillus subtilis strain FB17 root colonization on various upregulated and downregulated genes post FB17 colonization. For imaging, 15-d-old invitro grown seedlings of Col-0 and various mutants were rhizo-inoculated with FB17 or without FB17 (OD600 = 0.001). After 24 h of treatments roots were fixed in 4% formaldehyde formaldehyde solution solution and the roots were stained stained with SYTO13 (Invitrogen, Molecular Molecular Probes) to allow viewing of adherent FB17 cells on the root surface. Confocal fluorescence microscopy was performed as described by Lakshmanan et al.¹⁵ The strong green fluorescence along the sides of roots indicates root colonization of FB17. The bar indicates 50 μm.

Regulation of Bacillus subtilis FB17 colonization by Root Aluminum Activated Malate Transporter (ALMT1) expression

Recent studies showed that there is a positive correlation between B. subtilis root colonization, subsequent biofilm formation with enhanced local concentration of antibiotic concentration surrounding roots. The robust colonization is achieved by chemical communication between B. subtilis and plant host.^10,20 Plant root exudate and its composition is known to play an important role in plant-microbe interaction in the rhizosphere.^8,17,18,20 To investigate the cause of the profound colonization pattern of the FB17, the mutants of up-/downregulated genes were co-inoculated with foliar infiltration of PstDC3000 and root inoculated with FB17. Post 72 h of treatment FB17 colonization was quantified. Interestingly, post aerial treatment with PstDC3000 both mutants of up- and down- regulated genes showed significantly robust FB17 colonization. In addition, the increased FB17-root colonization was accompanied by activation of root aluminum activated malate transporter (ALMT1) expression. The results confirmed that PstDC3000 induced ALMT1 expression is independent and positive regulator of FB17 root colonization interaction.¹⁶ In addition, the data also shows that FB17 responsive genes may function independent of pathogen-induced ALMT1 expressions for FB17 recruitment.

Future perspectives

The signaling network between the host plants and rhizobacteria has been extensively studied over the past 20 years, a very few molecular components involved in the interaction between the host plant and the rhizobacteria had been reported until recently.^10,20 At this juncture, we may speculate that FB17 suppresses root innate immune system to establish the colonization. It will be interesting in the coming years to try and dissect the genetic component(s) of FB17 and its target on the host plants. The knowledge in this aspect will be instrumental in improving the agricultural application of FB17 as an agent for plant growth promotion and disease protection.

Lakshmanan V, Castaneda R, Rudrappa T, Bais HP. Root transcriptome analysis of Arabidopsis thaliana exposed to beneficial Bacillus subtilis FB17 rhizobacteria revealed genes for bacterial recruitment and plant defense independent of malate efflux. Planta. 2013;238:657–68. doi: 10.1007/s00425-013-1920-2.