Emerging Molecular Tools for Engineering Phytomicrobiome

Abstract

Microbial plant interaction plays a major role in the sustainability of plants. The understanding of phytomicrobiome interactions enables the gene-editing tools for the construction of the microbial consortia. In this interaction, microbes share several common secondary metabolites and terpenoid metabolic pathways with their host plants that ensure a direct connection between the microbiome and associated plant metabolome. In this way, the CRISPR-mediated gene-editing tool provides an attractive approach to accomplish the creation of microbial consortia. On the other hand, the genetic manipulation of the host plant with the help of CRISPR-Cas9 can facilitate the characterization and identification of the genetic determinants. It leads to the enhancement of microbial capacity for more trait improvement. Many plant characteristics like phytovolatilization, phytoextraction, phytodesalination and phytodegradation are targeted by these approaches. Alternatively, chemical communications by PGPB are accomplished by the exchange of different signal molecules. For example, quorum-sensing is the way of the cell to cell communication in bacteria that lead to the detection of metabolites produced by pathogens during adverse conditions and also helpful in devising some tactics towards understanding plant immunity. Along with quorum-sensing, different volatile organic compounds and N-acyl homoserine lactones play a significant role in cell to cell communication by microbe to plant and among the plants respectively. Therefore, it is necessary to get details of all the significant approaches that are useful in exploring cell to cell communications. In this review, we have described gene-editing tools and the cell to cell communication process by quorum-sensing based signaling. These signaling processes via CRISPR- Cas9 mediated gene editing can improve the microbe-plant community in adverse climatic conditions.

Introduction

Phytomicrobiome or plant microbiome represents the microbial communities that colonize with the various plant parts. These microorganisms include fungi, bacteria and archaea that collectively play a key role in the productivity, growth and health of the plant [1]. The phytomicrobiome may be rhizospheric (associated with roots), epiphytic (on the surface of plant parts) and endophytic (inner side of plant parts) [2]. It is reported that both soil microbes and mycorrhizal fungi can change the carbon composition into the soil by the exudation process of carbon. This exudation process increases the microbial activity and helps in the enhancement of more carbon decomposition. The rate of mineralization and soil decomposition increases by the interaction between microbes, mycorrhizal fungi and host plants [3]. A large number of inorganic and organic compounds synthesizes by microbes that help in encouraging root colonization. The microbial interaction can be of both types i.e. endophytic and epiphytic with their host plants. This type of interaction affects the plants either negatively or positively by several interactions such as amensalism, commensalism, and mutualism [4]. Endophytic bacterial colonization is the best example of microbe-plant interaction in which bacteria live within the host plant without causing any damage [5]. They colonize in the intercellular spaces and enter into the cell by cellulose degradation or local rupture of the roots. Different types of endophyte have been reported from different crops like cotton, sorghum, rice, wheat, maize, potato, sorghum and Arabidopsis. There are various studies conducted on microbe-plant interaction that express the regulation of cellular activity and signaling mechanisms [6, 7]. A better understanding of microbe-plant interaction can accomplish by various technique viz. gene-editing process and their cell to cell communication [8, 9]. Rhizospheric colonization exchanges different types of signals between bacteria and plants that can be beneficial, neutral or detrimental to the plants [10].

The ambition of phytomicrobiome engineering is to increase the significant outcomes of the plant including ISR, mineralization of organic matter and nutrient cycling during salinity and drought stress conditions [11]. The phytomicrobiome interactions depend upon various climatic conditions, soil type and plant species [12, 13]. Another major aspect of microbiome engineering is to harness all these variations during unfavorable conditions. This review also focuses on the several concepts of synthetic biology tools and their plant-microbe interactions by various signaling processes. By these approaches, stress response mechanisms during the biotic and abiotic conditions and their symbiosis process can also be understood.

Synthetic Biology for the Modification of Microbes

From the last few decades, various gene-editing tools have been designed and used for the enhancement of crop productivity by the modification of the local microbial community. Genetic engineering technique plays a major role in agriculture through recombinant DNA technology [14, 15]. The advancement of RNAi (RNA interference) has become the recent development in biotechnological genetic tools. It tends to the modification of targeted genes at the expression level. Along with RNAi, short-interfering and microRNAs (miRNA) also play a key role in gene expression. RNAi can manipulate and influence the essential traits of plants like branching, height, size and inflorescence. For example, gene OsDWARF4, can stimulate erect leaf structure in small plants by the RNAi manipulation process [16]. Also, the suppression of gene GA 20-oxidase (OsGA20ox2) can convert long rice (QX1 variety) into dwarf one. It was reported that the OsSPL14 gene is the target of miR156 gene in rice plants.The high rate expression of the OsSPL14 gene can lead to high grain yield with lower tiller number. Hence, miRNA and its targeted gene OsSPL14 are utilized for modifying the whole plant structure for the superior rice variety [17]. It is reported that the interaction of the OsmiR444a with D14 and OsMADS57 gene helps in controlling tillering in rice plants [18, 19]. The suppression of genes ipt and iaaM by RNAi mediated process can lead to the reduction in tumor formation in Agrobacterium tumefaciens. In Agrobacterium tumefaciens, the miR393 gene regulates TIR1 auxin receptors to reduce the infection by Pseudomonas syringae. Arabidopsis plants show high bacterial resistance by overexpression of miR393 gene [20]. Along with miR393, two other miRNAs, miR825 and miR398 cause a reduction in bacterial infections by down regulation. The expression of miR398 targets (CSD1 and CSD2) superoxide dismutases and causes up-regulation of bacterial infection under stress condition. The initiation process of RNAi occurs by the activation of ribonuclease protein Dicer [21]. After this process, some specific proteins bind with this siRNA and form a complex, which is integrated into RISC (RNA- induced silencing complex). The complementary mRNA sequence binds with the siRNA strand and results in the inhibition of the translation process and gene expression [22]. RNAi is used for the anti-pathogen activity in the agriculture field by the silencing process of targeted genes. A combinatory overview of modern technologies towards improving bioinoculants is illustrated in Fig. 1. It is reported that RNAi has the potential to engineer the local microbial community and provides resistance against pathogens. The gene-editing process becomes much easier with the development of CRISPR-Cas9 technology. During this process, the engineered and modified sgRNA (sequence-specific single guide RNA) forms a complex with Cas9. The genome of a cell can be edited with the removal or insertion on a specific location [23, 24]. Several reports have been demonstrated that the gene-editing approach helps in the enhancement of resistivity to various pathogens. By the approach of molecular tools, the desired phenotype and genotype can also be improved for the defence against pathogen and also for nutrient mobilization [11, 25, 26]. The information about some synthetic biology approaches and tools for improving microbial consortia is presented in Table 1. An assortment of recent approaches by gene-editing tools and their utilization method is depicted in Fig. 2. The engineering pathways help in the improvement of food production efficiency through the desired food ingredient. On the other hand, the engineering process involves those genes that led to the synthesis of unsaturated fatty acid by de novo process [36]. A lot of products that are traditionally derived from plants are now produced by engineered microbes. For example, resveratrol (heart health source) and stevia sweetener are now produced by a genetically engineered approach. The CRISPR-Cas9 technology, under synthetic biology, provides a better resistance mechanism against fungus and also responsible for turn off allergic reactions that affect the yield on large scale [37]. It is also reported that some aliphatic polyamines are found in microbes that help in the growth and development of plants. The most plentiful polyamines are spermidine and putrescine that play a major role in the signaling and differentiation of the cells [38, 39].

Fig. 1.

Combinatory overview of modern technologies towards improving bioinoculants

Table 1.

Synthetic biology tools and their subsequent approaches for improving microbial consortia

Synthetic biology tools	Microbes used	Synthetic biology approaches	Application	Other description	References
Engineered syntrophies and computational models	S. cerevisiae and E. Coli	Pathway separation, population control and DOL	Bioproduction of medicines, protein and biofuels	DOL, engineered signaling and pathway separation are the most usable approach	[27, 28]
Computational models and intercellular signaling	E. Coli	DOL and Spatial organization	Logic memory/computing	Logic gates are usually connected with signaling molecules and distributed between consortium members	[29, 30]
Engineered syntrophies, computational models and exogenous controllers	CoculturedS. Cerevisiae or E. coli with a microbe having activity against pollutant	Pathway separation	Deterioration of pollutants	Intensify the degradation process of pollutants, polymers and complex substrates	[31]
Exogenous controllers and intercellular signaling	B. subtilis and E. Coli	DOL	Biosensing	Detection of metabolites and small molecules	[32, 33]
Intercellular signaling and exogenous controllers	B. subtilis and E. Coli	DOL and Spatial organization	Functionalized biomaterials	Communication in synthetic consortia and production of functionalized biomaterials	[34, 35]

Fig. 2.

An assortment of recent approaches by gene editing tools and their utilization method in fields. In this a showing the strategies for improving microbial inoculants for formulation, b revealed the RNAi technology with the CRISPR/Cas method and c depicted the encapsulation process of microbes and their delivery method

Quorum Sensing and Communication Signals

In quorum sensing, the cell-cell communication process mediates by the tiny diffusible extracellular signaling molecules. In this process, gram-positive bacteria and gram-negative bacteria use peptide-signaling molecules and acylated homoserine lactones (AHLs) respectively that act as autoinducers [40]. These signals can activate various transcription factors at a specific population concentration of bacteria and help in the induction of genes in the bacterial community. Pantoea stewartii, Pseudomonas syringae and Pseudomonas aureofaciens rhizobacterial species utilize acylated homoserine lactones to control the microbial-plant interaction [41]. In Azospirillum lipoferum, Quorum sensing molecules are strain-specific and adapted to rhizospheric plant roots. Azospirillum brasilense supports both the process of chemokinesis and chemotaxis for chemo-effectors by enhancing the bacterial-root interaction. Microbial-plant interactions such as phytostimulation, biocontrol, biofertilization and rhizoremediation are QS dependent [42]. It is reported that various rhizobacterial species are capable of forming microcolonies on the roots of the plant and in induction of QS by high bacterial cell density. The secretion of mucigel by plant roots helps in the retardation of the diffusion of N-acyl homoserine lactones and in the coverage of bacteria which is required for quorum sensing. Mainly two N-acyl homoserine lactone systems RhlI/R and LasI/R are involved in the regulation of PGPR traits in Pseudomonas aeruginosa [43, 44]. Several health benefits could obtain by an effective signaling system [45, 46]. It is reported that root microflora and plants established a significant symbiotic relationship through a useful communication network [47, 48]. The rhizospheric plant roots are surrounded by an immense amount of microbial community that consists of hundreds to thousands of different species diversity [49]. Signaling pathways over the molecular level can control the microbial population in a better way by establishing a flourishing relationship between microflora and plants [50]. Many efforts have been made for knowing the molecular mechanism of communication network but still no adequate information to get the stability and evolutionary origins of the rhizosphere communication system. Table 2 represents the potential of gene editing tools for enhancing phytomicrobiome interactions. The association of Arbuscular mycorrhizal and PGPR-legume root symbioses existed by several signals of small diffusible molecules that is produced by both  microbe and plants. The root hairs provide a favorable biological surface for colonization, migration and chemotaxis to microbial communities [56]. A well-known symbiotic pathway is activated by signals that are produced by fungi and rhizospheric bacteria in the form of LCOs (lipo-chitooligosaccharides). These signals are understood via LysM (lysine-motif) that occur on the membrane of plants that help in the activation of CSP and regulation of rhizospheric microorganisms. It is reported that LysM receptor found in both non- legume plants and legume plants and receives signals from AM fungi (Myc-LCO signals) and rhizobia (nod factor signals) [5]. Numerous evidence demonstrated that the signaling pathway between roots of the host plant and AM fungi is much flourish and engaged for understanding the evolution of symbiosis [57]. Microbial communities and plants use various signaling pathways to convey useful information concerning their internal situation and colonization [3, 58]. In the symbiotic system of AM (arbuscular mycorrhiza) strigolactones molecule plays a major role. It is a plant hormone and terpenoid which is the byproduct of carotenoid metabolism [59]. It regulates the metabolic cycle of AM fungus and supports its growth towards the root [60]. Several types of strigolactones are produced by different plants for the attraction of AM fungi [61]. Strigolactones helps in the germination of arbuscular mycorrhiza fungal spores and bring out a series of signal molecule viz. lipo-chitooligosaccharides and chitooligosaccharides. These types of molecules trigger a set of various interactions in the plant root system. As a result of this, the cytosolic concentration of calcium increases and help in the induction of gene expression of mycorrhizal fungi that directs the penetration system. Plant roots promote the fungi for the initiation of arbuscular growth and hypopodium formation [62]. The plant hormone, auxin is synthesized by both PGPRs and plants via multiple pathways. Certain reports demonstrated that the IAA (indole-3-acetic acid) is a natural auxin that act as signaling molecules in the microbial community [63]. As a result of this IAA affects gene expression in several microbes and precedes reciprocal signaling molecule in microbe-plant interactions. IAA act as a controller of gene expression in IpdC gene of Azospirillum brasilense. IAA also has been observed as an inhibitory signaling molecule for the viral gene Agrobacterium tumefaciens [64]. Besides this, the level of auxin in microbe-plant interaction affects nodule formation and its differentiation [65]. Apart from this, chemical signaling has various consequences that affect microbe-plant interactions. The Als produced during interspecies communication can change the activities of its competitors. By disturbing this activity, beneficial microbes defend the host plant by inhibiting the activity of pathogens. In many cases, microbes produce VOCs and use enzymes like acylase and lactonase for the degradation of AHLs184 that disrupt bacterial AHL. Sometimes, microbes show a wide range of interactions from symbiotic to antagonistic and affect the immunity of plant [66]. Induced systemic resistance is controlled by these interconnected signaling pathways and their components. Plants frequently maintain both inducible and constitutive defense mechanisms by the process of hormonal signaling pathways. The phytohormones like JA, ethylene, auxins, ABA, SA and their derivatives play a vital role in the regulation of the immune system. According to the trophic relation of microbes, signaling molecules like JA and SA show a different type of immune response with their host plant. For instance, biotrophic microbes acquire nutritional requirements from living cells whereas necrotrophic obtain from dead plant cells by the production of phytotoxins. On the other hand, hemi-biotrophic microbes take nutrients from both types of cells i.e., from living or dead state. In hemi-biotrophic and biotrophic type only the SA defense pathway exists while in the necrotrophic JA pathway is effectual against pathogens. Consequently, signaling molecules enable both inter and intra microbial communication with the host plant and sustaining the colonization and symbiotic associations within plants [67].

Table 2.

Potential of gene editing tools for enhancing the phytomicrobiome interactions

Gene editing Tools	Traits present	Application	References
Cpf1 (gene replacement, gene knock-out, multiplex GE) and Cas9	Metabolic engineering	Exploration the metabolome pathways of novel plant	[51]
Cpf1 (gene replacement, gene knock-out, multiplex GE) and Cas9	Signaling molecules helps in Improvement of stress resistance	Enhances nutrients uptake	[52]
Cpf1 (gene replacement, gene knock in/out,) and Cas9	Improves nodulation in non-leguminous plants	Nutrient uptake and plant growth promotion	[52]
RNA editing tool and Cas13	In plant disease resistance	Identify pathogens	[53]
Base editing, Cpf1 (gene replacement, gene knock-out, multiplex GE) and Cas9	In plant disease resistance	Plant disease resistance against pest	[53]
EvolvR	Phytomicrobiome interactions	In allele generation	[54]
Lineage tracing and Genotyping	Phytomicrobiome interactions	In identification of genes that involves in phytomicrobiome interactions	[55]

Biofilm Production and Defence Induction by Microbes for Plant Growth

Biofilms are communities of microbial cells that are covered by extracellular polymeric substances (EPS). The formation of biofilm is initiated by the following factors like oxygen supply, osmolarity, pH and temperature [68]. Efficient root colonization by EPS-producing microbes helps in the circulation of essential nutrients to the host plant and prevents it attacking from foreign pathogens. Mainly PGPR is known for EPS characteristics that help in the formation of polymeric substances. Pseudomonas and Rhizobium species produce a variety of nucleic acids, organic molecules, proteins and polysaccharides that helps in the biofilm matrix. Biofilms developed in vitro are also used as biofilm biofertilizers. Furthermore, polysaccharides formed by rhizobial species also act as an active signal for defense response during any infection [69]. Many PGPR-EPS also bind Na⁺cations and help in the alleviation of salt stress by reducing Na⁺ content. It is also reported that the co-inoculation of biofilm microbial strain also helps in the induction of biocontrol mechanism.

Along with biofilm production, it is also reported that many species of microbes show endosymbiotically association and triggers the expression of plant genes that are responsible for different stress responses. Many studies showed that the Trichoderma that act as endosymbiont shows a good association with woody root plants and help in alleviating different abiotic stress condition and enhances the nitrogen-fixing efficiency of plants [70]. SA and JA mediated SAR and ISR responses are triggered by the higher dose of Trichoderma. Peptaibols and Xylanase are produced by various Trichoderma spp. that elicit a good immune response in plants and help in crop enhancement [71]. Recently, a hybrid enzyme polyketide synthases were identified in maize plants that involve in defence mechanisms. Epl1/Sm1 is an educer and secreted as cysteine-rich protein produced by Trichoderma spp .In maize crops, the deletion of the Trichoderma gene elicits the ISR response. On the other hand, it is also reported that a variety of secondary metabolites are produced by Trichoderma. Nonribosomal polyketides and peptides signify chief components of secondary metabolites. The pks4 gene from Trichoderma reesei was identified that involve in the synthesis of aurofusarin. The deletion of the gene in Trichoderma reesei results in the vanish of pigmentation and variation of teleomorphic structures. Besides this, the deletion of pks4 also influences the expression of PKS-encoding genes in Trichoderma reesei by affecting the stress resistance and defense mechanism.

Conclusion

Phytomicrobiome interactions play a significant role in the sustainability of plant health. These interactions result in beneficial, favorable or detrimental for plant growth and productivity. Hence, to control this complex process, signals or cell to cell communication play a crucial role between microbiome and plant. In this present review, gene-editing tools, cell-to-cell communication and their approaches have been described to understand the various factors that control microbiome congregation. Next step after this signaling process, new omics tools will certainly help in cultivating the microbiome for a better understanding of the engineering process. Furthermore, the biofilm production and polyketides gene identified in Trichoderma are also described with their application in defence induction for plant growth. By the engineering efforts, microbiome interactions will attain a sustainably, purposefully and presumably goal to engineer a better phytomicrobiome.

Compliance with Ethical Standards

Conflict of interest

The authors don’t have any conflict of interest.