Plant-Microbe Interaction and Genome Sequencing: An Evolutionary Insight

C H A P T E R

22 Plant-Microbe Interaction and Genome Sequencing: An Evolutionary Insight Krishna K. Sharma⁎, Deepti Singh⁎, Bijender Singh⁎, Sarvajeet S. Gill⁎, Amarjeet Singh†, Bhuvnesh Shrivastava‡ ⁎

Maharshi Dayanand University, Rohtak, India †University of Delhi, New Delhi, India ‡Panacea Biotech Limited, New Delhi, India

1 INTRODUCTION There is a global increase in demands for food, feed, and fuels, with limited availability of water and land resources (Sayer and Cassman, 2013). Among the biotic factors that limit our ability to overcome these challenges are the bacterial and eukaryotic filamentous plant pathogens that cause extensive annual yield losses of staple crops worldwide. The most devastating bacterial plant pathogen list includes (in rank order) (1) Pseudomonas syringae pathovars, (2) Ralstonia solanacearum, (3) Agrobacterium tumefaciens, (4) Xanthomonas oryzae pv. oryzae, (5) Xanthomonas campestris pathovars, (6) Xanthomonas axonopodis pathovars, (7) Erwinia amylovora, (8) Xylella fastidiosa (the causative agent of Pierce's disease on grapevines), (9) Dickeya (dadantii and solani), and (10) Pectobacterium carotovorum (and Pectobacterium atrosepticum). Other equally important plant pathogens are E. amylovora, Clavibacter michiganensis, C. michiganensis subsp. sepedonicus (Cms) (causal agent of ring rot of potato), and Erwinia chrysanthemi (Mansfield et al. 2012). Furthermore, ascomycete fungi, for example, rice-blast fungus Magnaporthe oryzae (Wilson and Talbot, 2009); the basidiomycete rust fungi (order Pucciniales) that plague several crop species (Duplessis et al., 2011); and oomycetes such as Phytophthora infestans (a potato late blight pathogen) have great impact on crop productivity (Vleeshouwers et al., 2011). However, there are opportunistic, avirulent plant fungal symbionts that establish robust and long-lasting colonizations of root surfaces and penetrate into the epidermal level. They release a variety of compounds that induce localized or systemic resistance responses, and

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22.  Plant-Microbe Interaction and Genome Sequencing: An Evolutionary Insight

this explains their lack of pathogenicity to plants. These root-microorganism associations cause substantial changes to the plant proteome and metabolism. Plants are protected from numerous classes of pathogen by responses that are similar to systemic acquired resistance and rhizobacteria-induced systemic resistance. For example, root colonization by Trichoderma spp. enhances root growth and development, crop productivity, resistance to abiotic stresses, and the uptake of essential nutrients (Harman et al., 2004). They are also effective biocontrol agents for numerous foliar and root phytopathogens, and some are also known for their abilities to enhance systemic resistance to plant diseases (Shoresh and Harman, 2008). Over the past 10 years, the genomes of more than 50 of these filamentous plant pathogens have been sequenced, revealing enormous diversity in genome size and sequence arrangement (Sharma, 2016). Coevolutionary conflicts with host plants have shaped the genomes of these filamentous plant pathogens into diverse architectures, but some common features can be noted in phylogenetically unrelated species (Raffaele and Kamoun, 2012). Genome studies have also demonstrated a concerted loss of genes for metabolism and hydrolytic enzymes and gain of diversity of genes coding for effectors involved in host defense suppression (Kemen et al., 2015). However, the identification of virulence targets for the hundreds of candidate effectors identified by genome sequencing remains a major challenge, partly because functional redundancy seems to be common and because high-throughput cell biological assays are lacking. An establishment of reliable transformation protocols for basidiomycetous fungi (Sharma and Kuhad, 2010) for obligate biotrophic rusts and mildews would facilitate studies of these systems, although new techniques such as host-induced gene silencing (HIGS) have also aided the analysis of effector candidates (Giraldo and Valent, 2013). A general scheme to understand microbial interaction with its host and environment is outlined in Fig. 1. Here, we highlight the genomic features of plant pathogens, with special emphasis on genome structure and plasticity. We also discuss the mechanisms and constraints that have driven the evolution of these genomes and the eventual virulence.

2  HOST-MICROBE AND MICROBE-MICROBE INTERACTION Microbial symbionts are associated with all plants in natural ecosystems having significant impact on plant communities through increasing fitness by conferring abiotic and biotic stress tolerance. The first description of symbiosis as “the living together of dissimilar organisms” (De Bary, 1879) and more than 135 years of research suggested that most, if not all, plants are symbiotic with mycorrhizal fungi and/or fungal endophytes affecting plant ecology, fitness, and evolution (Brundrett, 2006), shaping plant communities (Clay and Holah, 1999) and manifesting strong effects on the community structure and diversity of associated organisms including bacteria, nematodes, and insects (Omacini et  al., 2001) (Table  1). The fossil records suggested the association of plants with endophytic (Krings et al., 2007) and mycorrhizal (Redecker et  al., 2000) fungi for more than 400 million years, thus playing an important role in the evolution of life. Mycorrhizal fungi colonize only plant roots and grow into the rhizosphere, whereas endophytes reside entirely within plant tissues and may grow within stems, roots, and/or leaves (Stone et  al., 2004). There are two major groups of endophytic fungi, namely, the clavicipitaceous endophytes (C-endophytes) and nonclavicipitaceous endophytes (NC-endophytes), exhibiting differences in evolutionary relatedness,



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Plant-associated microbes (Viruses, bacteria, fungi, oomycetes, and nematodes etc)

Standardized bioinformatics tools Integrate, manipulate, analyze, compare, data generated by structural and functional analysis Flexible (comparable among different genomes, including microbes and plants) Scalable and inter-operable, readily Gene libraries, and genetic maps

Structural analysis Key to gene discovery and point of initiation

Functional analysis Gene expression and proteomic analyses for functional identification of genes and proteins involved in interactions of microbes with plants and environment. Array-based technologies Metatranscriptomics approaches High-throughput genome-wide gene deletion and tagging

FIG. 1  A systematic integrated approach to study microbial genome-mediated plant-microbe interaction.

TABLE 1  Fungal Endophytes Confirming Biotic and Abiotic Stress Tolerance S. No. Endophyte

Stress

Host

Reference

1.

Neotyphodium tembladerae

Mammals

Grasses

Gentile et al. (1999)

2.

Neotyphodium gansuense

Animals

Achnatherum inebrians

Li et al. (2004)

3.

Epichloë festucae

Fungi

Turfgrasses

Clarke et al. (2006) and Bonos et al. (2005)

4.

Curvularia protuberata

High temperature

Dichanthelium lanuginosum

Márquez et al. (2007)

5.

Fusarium culmorum

Salinity

Leymus mollis

Rodriguez et al. (2008)

6.

Fusarium oxysporum

Disease resistance

Hordeum vulgare

Schulz et al. (1999)

7.

Curvularia protuberate

Drought

Oryza sativa

Rodriguez et al. (2008)

8.

Curvularia protuberata

Heat

Lycopersicon esculentum

Rodriguez et al. (2008)

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taxonomy, host plants, and ecological functions. C-endophytes infect some grasses, while NC-endophytes inhabited asymptomatic tissues of nonvascular plants, ferns and allies, conifers, and angiosperms.

2.1 C-Endophytes They are also called as class 1 endophytes, representing a small number of phylogenetically related clavicipitaceous species that are fastidious in culture and limited to some cool- and warm-season grasses (Bischoff and White, 2005). These endophytes occur within plant shoots and form systemic intercellular infections. Clay and Schardl (2002) recognized three types of clavicipitaceous endophytes, ranging from symptomatic and pathogenic species (Type I) to mixed interaction (Types II) and asymptomatic endophytes (Type III). Transmission of these endophytes is vertical, that is, colonized plants pass endophytes to their offspring by seed infections (Saikkonen et al., 2002). These endophytes increase plant biomass, confer drought tolerance, and decrease herbivory by producing toxic compounds. However, these benefits are dependent on the host species, host genotype, and environmental conditions (Faeth et al., 2006).

2.2 NC-Endophytes They are highly diverse and ubiquitous microorganisms comprising of a polyphyletic assemblage of primarily ascomycetous fungi. These endophytes have been recovered from every major lineage of land plants and from all terrestrial ecosystems, including both agroecosystems and biomes ranging from the tropics to the tundra (Arnold and Lutzoni, 2007). NC-endophytes are divided into three functional classes on the basis of host colonization patterns, mechanism of transmission between host generations, in planta biodiversity levels, and ecological function. Among these, class 2 endophytes may grow in both above- and belowground tissues, while class 3 and 4 endophytes are confined to aboveground tissues and roots, respectively. Colonization of host tissues also differs as class 3 endophytes form highly localized infections, while class 2 and 4 endophytes show extensive tissue colonization. Class 2 endophytes also are transmitted horizontally and vertically, while class 3 endophytes are transmitted only in horizontal manner. Class 2 endophytes also confer habitat-specific stress tolerance such as pH, temperature, and salinity to host plants (Rodriguez et al., 2008). Among all the classes, only class 2 endophytes have been shown to confer habitat-adapted stress tolerance (Rodriguez et al., 2008).

3  CLAVICIPITACEOUS ENDOPHYTES AND THEIR ROLE These endophytes of grasses have been reported in the seeds of Lolium temulentum, L. arvense, L. linicolum, and L. remotum (Rodriguez et al., 2009). Most clavicipitaceous endophytes are known to enhance resistance of hosts to insect feeding (Rodriguez et  al., 2009). Tintjer and Rudgers (2006) found that fungal strain and growth stage of the plant are responsible for insect herbivory. However, there is contradiction regarding the insect or nematode resistance provided by class 1 endophytes. Kimmons et  al. (1990) have shown antinematode activity



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of class 1 endophytes, while Faeth et al. (2006) did not observe such type of activity. Some endophytes protect plants from feeding by mammalian herbivores (Li et al., 2004). For example, sleepy grass (Achnatherum robustum), common in the western United States, harbors an endophyte that produces lysergic acid amide. Horses that consume relatively small quantities of infected grass will sleep for up to 3 days and then gradually recover. Next time, animals avoid eating of these plants. In South America, Neotyphodium tembladerae infects several species of grasses that are reported toxic to the mammals (Gentile et al., 1999). In Asia, drunken horse grass (A. inebrians) is infected by N. gansuense and similarly avoided by animals (Li et al., 2004).

4  EFFECTS ON DISEASE RESISTANCE AND SUSCEPTIBILITY There are very few studies regarding the effects of class 1 endophytes on disease resistance. These endophytes produce compounds that inhibit the growth of other fungi. Yue et al. (2000) identified several indole derivatives, a sesquiterpene, and a diacetamide from Epichloë festucae. In the case of E. festucae, infected turfgrasses have shown significant resistance to two major leaf-spot pathogens Sclerotina homeocarpa (causing dollar spot disease) (Clarke et al., 2006) and Laetisaria fuciformis (causing red thread disease) (Bonos et al., 2005).

5  ROLES IN PLANT ECOPHYSIOLOGY Class 1 endophytes enhanced the ecophysiology of host plants by enabling them to counter abiotic stresses such as drought (Arechavaleta et al., 1989) and metal contamination (Malinowski and Belesky, 2000). Malinowski and Belesky (2000) observed that N. coenophialum infection resulted in the development of extensive root systems that enable plants to better acquire soil moisture and absorb nutrients under stress conditions. These endophytes resulted in the formation of longer root hairs. These roots hairs secrete phenolic-like compounds into the rhizosphere, which is responsible for efficient absorption of soil phosphorus and enhanced aluminum tolerance via chelation (Malinowski and Belesky, 2000). Zaurov et al. (2001) artificially infected several fine fescue clones with Neotyphodium sp. and found that some combinations had a negative effect on plant mass, some were neutral, and others increased plant biomass. Similarly, some combinations enhanced tolerance to soil aluminum; others had no effect or reduced tolerance compared with endophyte-free clones (Zaurov et al., 2001). This study demonstrates that genotype-specific interactions may enhance, reduce, or have no effect on plant fitness.

6  CLASS 2 ENDOPHYTES AND THEIR ROLES Class 2 endophytes comprise a diversity of species, all of which are members of the Ascomycota and Basidiomycota. Members of Ascomycota are restricted to the Pezizomycotina, while Basidiomycota includes the members from the Agaricomycotina and Pucciniomycotina. Class 2 endophytes are different from the other NC-endophytes as they colonize roots,

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stems, and leaves forming extensive infections within plants and are transmitted via seed coats and/or rhizomes. They provide habitat-adapted fitness benefits to plants in addition to nonhabitat-adapted benefits. Plants growing under high-stress habitats are highly infected with these endophytes.

7  SYMBIOTICALLY DERIVED BENEFITS TO ENDOPHYTES Some endophytes avoid stress through plant symbiosis. For example, Curvularia protuberata colonizes all nonembryonic tissues of the geothermal plant Dichanthelium lanuginosum (Redman et al., 2002; Márquez et al., 2007). When grown nonsymbiotically, neither the plant nor the fungus can tolerate temperatures above 40°C. However, the symbiosis allows both partners to tolerate temperatures up to 65°C. Similarly, Fusarium culmorum forms symbiosis with coastal dune grass (Leymus mollis) and tolerate salinity up to 500  M NaCl, but when grown nonsymbiotically, both fail to grow under this salinity stress (Rodriguez et al., 2008). This clearly indicates that both endophytes negate deleterious effect of stresses (temperature and salt) by colonizing in plant tissues. Class 2 endophytes exhibit mutualistic interaction, conferring positive fitness benefits to hosts and also obtaining nutrition for growth and reproduction from host tissues and avoiding abiotic stress via symbiosis.

8  ENDOPHYTE-CONFERRED FITNESS BENEFITS AND ECOLOGICAL ADAPTATIONS OF PLANTS Most of the class 2 endophytes have shown improvement in host biomass either due to the induction of plant hormones by the host or biosynthesis of plant hormones by the endophytes (Tudzynski and Sharon, 2002). Many class 2 endophytes protect host plants against fungal pathogens (Campanile et al., 2007). Endophytic isolates of F. oxysporum and a Cryptosporiopsis sp. conferred disease resistance against virulent pathogens in barley (Hordeum vulgare) and larch (Larix decidua), respectively, due to the production of phenolic metabolites (Schulz et al., 1999). Class 2 endophytes are unique in their ability to colonize asymptomatically and provide habitat-adapted fitness benefits on genetically distant host species representing monocots and eudicots (Rodriguez et al., 2008). Research studies have shown that C. protuberata provides heat but not salt or disease tolerance, F. culmorum shows salt but not heat or disease tolerance, and Colletotrichum spp. provides disease resistance but not heat or salt tolerance (Redman et al., 2002; Rodriguez et al., 2008). Additional studies revealed that the ability of endophytes to confer habitat-specific stress tolerance is an adaptive process defined at the subspecies level (Rodriguez et  al., 2008). All of these endophytes establish nonpathogenic symbioses, but the fitness benefits are dependent on the habitat-specific stresses. All of these endophytic fungi provide drought tolerance and growth enhancement on various host species (Rodriguez et al., 2008), indicating that they were expressing mutualistic lifestyles. These results suggest that the symbiotic associations required for stress tolerance predate the divergence of these plant lineages between 140 and 235 million years ago (Chaw et al., 2004). The ability of endophytic fungi (e.g., C. protuberata, F. culmorum, M. ascophylli, and Colletotrichum spp.) to provide drought tolerance is responsible for the movement of plants onto land (Pirozynski and Malloch, 1975).



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9  MECHANISMS OF STRESS TOLERANCE Class 2 endophytes generally increase plant biomass under stress conditions, but the exact mechanism for this process is still unexplained. Endophytes activate host defenses more rapidly after infection with pathogens as compared with nonsymbiotic plants (Redman et  al., 1999). Stress conditions like heat, drought, and salt altered water relations and increased osmolyte production, production of signaling molecules such as abscisic acid (ABA), and the generation of reactive oxygen species (ROS) (Wang et al., 2003; Apel and Hirt, 2004). When plants are exposed to abiotic stress of paraquat (a herbicide that induces ROS production and subsequent photobleaching of chlorophyll), nonsymbiotic plant tissues bleach white, while symbionts (class 2 endophyte-colonized) plant tissues remain green (Rodriguez et al., 2008). Drought tolerance in plants is often correlated with increased osmotic potential (Wang et al., 2003). However, class 2 endophyte-conferred drought tolerance does not correlate with an increase in osmotic potential (Rodriguez et al., 2008). Remarkably, symbiotic plants consume less water (30%–50%) than nonsymbiotic plants regardless of the class 2 endophyte, suggesting that water usage efficiency may be more important for drought tolerance than osmolyte modulation. Class 2 endophytes significantly affect the ecophysiology of plants resulting in rapid adaptation of plants under stress conditions.

10  CLASS 3 ENDOPHYTES Class 3 endophytes form localized infections in aboveground tissues showing horizontal transmission and high in planta biodiversity. They include the hyperdiverse endophytic fungi associated with leaves of tropical trees (Arnold et  al., 2000; Gamboa and Bayman, 2001) and the highly diverse associates of aboveground tissues of nonvascular plants, seedless vascular plants, conifers, and woody and herbaceous angiosperms in biomes ranging from tropical forests to boreal and Arctic/Antarctic communities (Higgins et al., 2007; Murali et al., 2007; Davis and Shaw, 2008). They are also found in flowers and fruits and in asymptomatic wood and inner bark (Tejesvi et al., 2005). Individual plants may harbor hundreds of species, and plant species across their native ranges may be inhabited by thousands of species. There is great diversity in class 3 endophytes infecting a large range of plants and tissues. Plants infected with these endophytes do not show observable change in growth rate, biomass accumulation, root-shoot ratio, and other easily quantifiable characteristics following inoculation under in vivo conditions (Arnold et al., 2003a,b). There are distinct observations regarding their effects on plants. Schulz et al. (1999) observed negative effect of class 3 endophytes on plant growth, while Arnold and Engelbrecht (2007) demonstrated that some seedlings lose water more quickly under drought stress infected with class 3 endophytes. Class 3 endophytes may play major roles in the interaction of other organisms with plants. Several studies have pointed to the diverse ecological roles and potential applications of class 3 endophytes—a hyperdiverse group that remains woefully understudied (Arnold et al., 2003a,b; Arnold and Engelbrecht, 2007).

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11  CLASS 4 ENDOPHYTES Merlin (1922) during the isolation of ectomycorrhizal fungi observed a brown to blackish, pigmented fungus associated with terrestrial plant roots. He called these sterile, root-­ associated fungi “mycelium radicis atrovirens” (MRA). MRA were often found to coexist with mycorrhizal fungi and were referred to as “pseudomycorrhizal” fungi (Merlin, 1922). Shortly thereafter, Peyronel reported  >  135 species of angiosperms associated with dark-­ pigmented fungi in root tissues (Peyronel, 1924). Currently, these fungi are collectively called as “dark septate endophytes” (DSE), grouped together as class 4 endophytes. These endophytes are distinguished as a functional group based on the presence of darkly melanized septa and their colonization with plant roots only. Class 4 endophytes are conidial or sterile ascomycetous fungi that form melanized structures such as inter- and intracellular hyphae and microsclerotia in the roots. These endophytes are prevalent in high-stress environment and are ubiquitous in occurrence ranging from Antarctic, Arctic, alpine, subalpine, and temperate zones, as well as from African coastal plains and lowlands and some tropical ecosystems (Jumpponen and Trappe, 1998). These fungi are nonpathogenic as observed on healthy fine roots causing no adverse effects (Jumpponen and Trappe, 1998). There is very scanty information regarding the role of class 4 endophytes. Mandyam and Jumpponen (2005) have proposed that endophyte colonization may play a role in deterring pathogens by minimizing available carbon in the rhizosphere and that high melanin levels may potentially be involved in the production of secondary metabolites toxic to herbivores, both factors giving class 4 symbiotic plants a competitive edge. The role of class 4 endophytes must indeed be important in plant ecophysiology beside their less information.

12  HOST-MICROBE INTERACTION AND PLANT IMMUNITY Both aerial and terrestrial microbes have evolved to colonize the plants for their mutual benefits. The niches of the microorganisms may be epiphyte (present on the surface of the plant), endophyte (found inside the plant tissue), phyllospheric (growing on leaf surface), and rhizospheric (inhabiting soil closely associated with roots). Out of these diverse niches, rhizosphere is the most dynamic due to its massive impact on plant nutrition, growth, and evolution (Raffaele and Kamoun, 2012; Kemen et  al., 2015; Bandyopadhyay et  al., 2016). Numerous bacterial and filamentous eukaryotes (fungi and oomycetes) have adopted an obligate parasitic lifestyle on plants in which they interact and coevolve with their host (Alfano and Collmer, 2004; Kemen et al., 2015). Fungal pathogens grow asymptomatically for long periods; however, the symptoms occur only when spores are released during the reproductive phase, either by rupturing the epidermis or reproductive structures, such as conidiophores, that grow on the leaf surface or come out through stomatal openings (Kemen et al., 2015). Earlier workers have shown that different species of Trichoderma are opportunistic fungi, avirulent plant symbionts, and very common in soil and root ecosystems (Harman et  al., 2004). At least some strains establish robust and enduring colonization of root surfaces, which later penetrate into the epidermis and a few cells below. They produce or release a variety of compounds that induce localized or systemic resistance responses, and this explains



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their lack of pathogenicity to plants. The root colonization by Trichoderma spp. also enhances root growth and development, shoot growth, crop productivity, resistance to biotic and abiotic stresses, and the uptake of nutrients from the soil. Also, plants are protected from numerous plant pathogens by responses that are similar to systemic acquired resistance and rhizobacterium-induced systemic resistance (Harman et al., 2004). These root-microorganism associations result in substantial changes to the plant proteome and metabolism. Interestingly, the genomes of many parasites and bacterial symbionts have been significantly reduced over time; the genomes of several lineages of filamentous plant pathogens have been shaped by repeat-driven expansions (Raffaele and Kamoun, 2012). Piriformospora indica, an axenically cultivable phytopromotional, biotrophic mutualistic root endosymbiont that belongs to the order Sebacinales (Basidiomycota), colonizes roots of a wide range of higher plants and provides multifaceted amenities (such as nutrient uptake, disease resistance, stress tolerance, and growth promotion involving value addition) to plants (Unnikumar et al., 2013; Gill et al., 2016). P. indica has been extensively reported to improve crop tolerance to a number of abiotic stresses including salinity, low temperature, and heavy-metal toxicity (Baltruschat et al., 2008; Sun et al., 2010; Ansari et al., 2013; Unnikumar et al., 2013). P. indica colonization has extensively been reported to mediate the activation of abiotic stress-responsive genes (DREB2A, CBL1, and RD29A) (Ansari et  al., 2013) and osmoprotectants (proline and glycine betaine) (Waller et al., 2005; Trivedi et al., 2013). The interaction of P. indica with A. thaliana roots is a unique model system to study symbiotic relationships. Recently, Vahabi et al. (2015) has reported a cocultivation system that allowed them to investigate the effects of fungal exudates on the root transcriptome before and after the establishment of a physical contact and during early phases of root colonization. Plants have evolved sophisticated mechanisms to perceive pathogen attack and trigger an effective innate immune response. An important and well-characterized perception mechanism is based on resistance (R) genes in plants whose products confer recognition of cognate avirulence (AVR) proteins in the pathogen (van der Hoorna and Kamoun, 2009). Filamentous eukaryotic pathogens of cultivated crops remain major food-security threats, and the incorporation of R genes into these crops provides the best solution for sustainable disease control. High-throughput effectoromics methods (a functional genomics approach that uses effectors to probe plant germplasm to detect R genes) for screening the large sets of effector gene candidates available from genome sequencing are already being used to identify new avirulence effectors (AVR) gene-R gene pairs that might be useful to agriculture (Vleeshouwers et al., 2011; Stergiopoulos and de Wit, 2009). Many phytopathogenic bacteria inject virulence effector proteins directly inside the plant cells via a hypersensitive response and pathogenicity (Hrp) type-III secretion system (T3SS) (Nans et al., 2015). Pathogens are dependent on T3SS for defeating basal defenses and thereafter grow into the plant's tissue to produce disease lesions in hosts and elicit the hypersensitive response (HR) in nonhosts. The Hrp T3SS employs customized cytoplasmic chaperones, conserved export components in the bacterial envelope (also used by the T3SS of animal pathogens), and a more specialized set of T3SS-secreted proteins to deliver effectors across the plant cell wall and plasma membrane (Alfano and Collmer, 2004). In P. indica, the evidence of biotroph-associated genomic adaptations can be observed, where the genes involved in N metabolism are lacking and also a limited potential is d ­ isplayed

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by this mutualistic symbiont for host damage and destruction (Zuccaro et al., 2011). In fact, P. indica lacks genes potentially involved in the biosynthesis of toxic secondary metabolites and cyclic peptides. Nevertheless, genomic traits with saprotrophic and hemibiotrophic phytopathogenic fungi (such as the presence of an expanded enzyme arsenal that is weakly expressed during the initial biotrophic phase) are also shared by P. indica (Zuccaro et al., 2011). The analyses of the colonized young H. vulgare roots have revealed 579 genes in the phase of prepenetration (36–48 hpi), 397 genes at early colonization (3 dpi), and 641 genes (5 dpi) as distinctively regulated when compared with fungal-free roots (Zuccaro et al., 2011). In addition, the majority of genes (40%) induced via symbiosis were found to be nonorthologous to either species; rather, these were reported specific to P. indica (Zuccaro et al., 2011). Plant hormones, namely, jasmonic acid (JA), methyl jasmonate, and ethylene signals from the roots, were reported to decide for the shoots to become preconditioned prior to foliar pathogen infection in P. indica-colonized roots via activating defense responses that in turn were evidenced to lead to enhanced disease resistance (Stein et al., 2008). In a signaling cascade, the JA (VSP, PDF1.2, and LOX2) plus ET (ERF1) signaling and not the SA signaling (PR1, PR5) genes were upregulated in the P. indica plants to cope the powdery mildew fungus (Waller et al., 2005; Unnikumar et al., 2013). The indole-3-acetic acid (IAA) and ethylene role have been implicated in establishing a biotrophic symbiosis representing a compatibility factor at the contact surface between endophyte and plants (Hilbert et al., 2012; Khatabi et al., 2012). The augmented intracellular Ca2 + pool after attaining the basic compatibility between the two partners in an early signaling event in the interaction of endophyte with plants (McAinsh and Pittman, 2009) acts as a second messenger in various plant signaling pathways (Sanders et al., 2002; Ramakrishna et al., 2016). Recently, Kogel and coworker reported the function of P. indica effector candidate PIIN_08944, a non-DELD effector, during the interaction of plants with P. indica. The candidate effector was found to contribute in plant colonization by the mutualistic fungus by suppressing the salicylate-mediated basal resistance response (Akum et al., 2015). Effector protein, PIIN_08944 expression, was detected during chlamydospore germination, and fungal deletion mutants (Pi∆ 08944) showed delayed root colonization. Moreover, PIIN_08944expressing A. thaliana has showed a reduced expression of flg22-induced marker genes of pattern-triggered immunity (PTI) and the salicylic acid defense pathway. In barley, the expression of PIIN_08944 reduces the burst of reactive oxygen species (ROS) triggered by flg22. Therefore, the effector PIIN_08944 contributes to root colonization by P. indica by interfering with salicylic acid-mediated basal immune responses of the host plant (Akum et al., 2015).

13  MICROBIAL GENOME AND PLANT-MICROBE INTERACTION The first batch of genome sequences of plant pathogenic fungi and oomycetes marked the emergence of a new research field centered on the genome biology of these important pathogens. The genome sequences have revealed a lot of new information about the evolution of these fascinating microorganisms and the genomic features that underlie their success. Most strikingly, several lineages of filamentous plant pathogens, particularly the biotrophs, are remarkable among pathogenic organisms in displaying an evolutionary trend toward bigger,



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transposable element-rich genomes (Raffaele and Kamoun, 2012). Now, the genome-based studies on plant-associated microorganisms have changed our understanding of plant pathogens and also transformed our knowledge of mutualistic and commensal interactions with economically important plants (Guttman et al., 2014). In filamentous fungi, the effector genes are also commonly found in association with rapidly evolving segments of the genome, such as repeat-rich regions or on accessory chromosomes (ACs). For example, AvrPita in M. oryzae, SIX genes in F. oxysporum, and the PEP cluster in Nectria haematococca are all located on ACs (Rodriguez-Carreset al., 2008). Zymoseptoria tritici has several ACs that are well described, though, unlike other fungal pathogens, they have never been associated with pathogenicity (Croll et al., 2013). Other well-­characterized necrotrophic effectors, such as ToxA in Pyrenophora tritici-repentis and Tox3 and Tox1 in Parastagonospora nodorum, were successfully identified using culture filtrates that induced necrosis when infiltrated into susceptible wheat varieties (Liu et  al., 2012). This approach has recently identified two necrosis-inducing proteins, ZtNIP1 and ZtNIP2, in Z. tritici (Ben M'barek et al., 2015). The heterologous expression and infiltration of these proteins into wheat also revealed cultivar specificity (Ben M'barek et al., 2015). Recently, Solomon and coworkers have developed a gene tree sorting method that quickly identifies groups of isolates within a single gene alignment whose sequence haplotypes correspond with virulence scores on a single wheat cultivar (Z. tritici) (McDonald et al., 2016). Using this method, they have identified 100 candidate effector genes whose gene sequence correlates with virulence toward a wheat cultivar carrying a major resistance gene. Earlier, several in-depth RNA-sequencing (RNA-seq) studies with Z. tritici have identified many highly expressed “effector-like” genes or secondary metabolite clusters; however, no effector genes critical for virulence were identified in these studies (Kellner et al., 2014; Rudd et  al., 2015). Thus far, the only gene that has been shown to be essential for virulence in Z. tritici is Mg3LysM, which was discovered based on close homology to another previously described effector gene Ecp6 (Marshall et al., 2011). Three additional small secreted proteins (SSPs) that contribute quantitatively to virulence were recently described by Poppe et al. (2015). These genes were selected for functional analysis because they exhibited positive (syn. diversifying) selection (dN/dS > 1), when compared with genomes of nonwheat-infecting relatives Zymoseptoria pseudotritici and Zymoseptoria ardabillae (Poppe et al., 2015). Pathogen-genome projects employing bioinformatics methods to identify T3SS-Hrp regulon promoters and T3SS-pathway targeting signals suggest that phytopathogenic Pseudomonas, Xanthomonas, and Ralstonia spp. harbor large arsenals of effectors (Alfano and Collmer, 2004). T3SS effectors are commonly associated with mobile genetic elements, and many appear to have been acquired by horizontal gene transfer (Arnold et al., 2003a,b). For example, the P. syringae effector genes are associated with regions missing in P. aeruginosa and P. putida, and some are carried on plasmids or in exchangeable effector locus, which is a hypervariable region of the genome (Alfano et  al., 2000; Deng et  al., 2003; Buell et  al., 2003; Alfano and Collmer, 2004), whereas the exchangeable effector locus in P. syringae (e.g., hypervariable region) and another apparent hot spot for effector gene recombination have been identified at the different region of the genome (Alfano et al., 2000). Furthermore, the genomes that have been sequenced to date, the hypersensitive response and pathogenicity (hrp) or hypersensitive response and conserved (hrc) genes, are found clustered in a single r­ egion

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of the chromosome or on a 2.1 Mb megaplasmid in the case of R. solanacearum or a 150 kb plasmid in Pantoea agglomerans (E. herbicola) pv. gypsophilae (Alfano and Collmer, 2004). These clusters of hrp/hrc genes are typically flanked by regions that contain different effector genes in different bacterial species or pathovars (Alfano et al., 2000; Alfano and Collmer, 2004). P. syringae is best known as a plant pathogen, causing disease by translocating immune-­ suppressing effector proteins into plant cells through a T3SS (Zhou and Chai, 2008). However, P. syringae strains belonging to a newly described phylogenetic subgroup (group 2c) are missing the canonical P. syringae hrp/hrc cluster coding for a T3SS, flanking effector loci, and any close orthologue of known P. syringae effectors (Clarke et al., 2010). Nonetheless, P. syringae group 2c strains are common leaf colonizers and grow on some tested plant species to population densities higher than those obtained by other P. syringae strains on nonhost species. Moreover, group 2c strains have genes necessary for the production of phytotoxins, have an ice nucleation gene, and, most interestingly, contain a novel hrp/hrc cluster, which is only distantly related to the canonical P. syringae hrp/hrc cluster. This hrp/hrc cluster appears to encode a functional T3SS although the genes hrpK and hrpS, present in the classical P. syringae hrp/hrc cluster, are missing. The genome sequence of a representative group 2c strain also revealed distant orthologues of the P. syringae effector genes avrE1 and hopM1 and the P. aeruginosa effector genes exoU and exoY (Clarke et al., 2010). However, the draft genome sequence of P. syringae Psy642 revealed an atypical hrp/hrc region inserted in a different genomic location compared with the conserved location of the canonical P. syringae hrp/hrc cluster. This hrp/hrc cluster was found to be located between the orthologues of two neighboring PsyB728a genes, Psyr_1587 and Psyr_1588, which code for a bile acid/sodium symporter and a recombination associated protein, respectively. Furthermore, in the completely sequenced genomes of P. syringae (strain PtoDC3000 and Pph1448A), these two genes flank each other (Clarke et al., 2010).

14  LIFESTYLE TRANSITION IN PLANT PATHOGENS Major hemibiotrophic plant pathogens such as Colletotrichum and the rice-blast fungus M. oryzae undergo major transformations in cell morphology and infection mode when switching from growth on the plant surface to intracellular biotrophy and from biotrophy to necrotrophy. Genome sequencing combined with high-throughput transcriptome sequencing revealed the transcriptional dynamics underlying these transitions and led to redefine the functions of appressoria and intracellular hyphae (O'Connell et al., 2012). Further, comparative genomics showed that fungi have large sets of pathogenicity-related genes, but families of genes encoding secreted effectors, pectin-degrading enzymes, secondary metabolism enzymes, transporters, and peptidases are expanded in Colletotrichum higginsianum. The genus Colletotrichum (Sordariomycetes, Ascomycota) comprises ~ 600 species and attacks and colonizes on approximately 3200 species of monocot and dicot plants (ARS Fungal Databases). O'Connell and coworkers had sequenced two Colletotrichum species with different host specificities and infection strategies: C. higginsianum attacks several members of Brassicaceae, including Arabidopsis, and has emerged as a tractable model for studying fungal pathogenicity and plant immune responses (O'Connell et al., 2004, 2012). In contrast, C. graminicola primarily infects maize (Zea mays); biotrophy extends into many host cells and persists at the advancing colony margin, while the center of the colony becomes necrotrophic (O'Connell et al., 2012).



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The genome and transcriptome of C. higginsianum infecting A. thaliana and C. graminicola infecting Z. mays have been analyzed. Comparative genomics showed that both fungi have large sets of pathogenicity-related genes, but the families of genes encoding secreted effectors, pectin-degrading enzymes, secondary metabolism enzymes, transporters, and peptidases are expanded in C. higginsianum. Furthermore, genome-wide expression profiling revealed that these genes are transcribed in successive waves that are linked to pathogenic transitions: effectors and secondary metabolism enzymes are induced before penetration and during biotrophy, whereas most hydrolases and transporters are upregulated later, at the switch to necrotrophy (O'Connell et al., 2012).

15  GENOME EVOLUTION IN BACTERIAL AND FUNGAL PLANT PATHOGEN Pathogenic fungi and bacteria can lead to severe economic losses due to infected crops; therefore, it is of great concern to food security. The increasing global transportation of plant and plant products creates new combinations of their associated pathogens. Such events need serious attention because they may lead to the emergence of diseases with new epidemiological properties or host specificities (Brasier, 2001). Further, hybridization events have also given rise to a variety of genomic constitutions and evolutionary consequences (Mallet, 2007). Compared with animals and plants, the genome of fungi exhibits gene-dense genomes, with an average estimated size of ~ 37  Mb and ranging between 6.5  Mb for Pneumocystis carinii and 795  Mb for Scutellospora castanea (Gregory et  al., 2007). There is also significant variation in chromosome numbers in fungi, with the smallest number of 3 in the ascomycete Schizosaccharomyces pombe and the largest number of 20 in the basidiomycete Ustilago hordei and the chytrid Batrachochytrium dendrobatidis (Gregory et al., 2007). Genome evolution has taken place mainly by three main forces, that is, gene gain, gene loss, and gene change. Comparative genomics showed that fungi and bacteria have different modes of host adaptation on the genomic level. The pathogenic lifestyle of fungi suggests the tendency for reduced genome size in fungi (Yuen et al., 2003). This signature of adaptation can be acquired either by losing genes or whole metabolic pathways that are no longer necessary; for example, Hemiascomycetes have lost the genes needed to survive on the carbon source galactose that was irrelevant within a new host environment (Hittinger et al., 2004). In spite of common themes in fungal evolution, fungi are strikingly diverse at the genome level and mostly showing lineage-specific evolution. They not only are highly divergent in DNA sequences but also are striking changes in the order and localization of homologous genes among genomes. For example, the comparison of ascomycetes Neurospora crassa and Magnaporthe grisea reveals that their genomes have only 74% identity at the amino acid level and with virtually no similarity between the chromosomal fragments (Dean et  al., 2005). Furthermore, most pathogenic fungi have also experienced the expansion of specific gene families related to functions that facilitate the infection of the host. An example of how the expansion of specific gene families provides pathogenic potential to an organism is given by the genome of Penicillium marneffei, the only known pathogenic fungus of the Penicillium genus. Compared with its progenitors and relatives, P. marneffei has adopted reductive genome evolution (17 Mb compared with ~ 30 Mb in other Penicillium species), and its genome is rich

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in secondary metabolite genes and thioester-mediated nonribosomal protein synthesis (Yuen et  al., 2003). Another fungus showing peculiar genomic features related to its pathogenic lifestyle is Ustilago maydis, a biotroph basidiomycete that parasitizes maize and depends on living tissue for proliferation and development. Not surprisingly, it lacks the pathogenicity genes present in more aggressive necrotrophic fungal pathogens. However, it possesses a cluster of secreted protein effectors that favors the invasion of living tissue and eventually minimizes host damages (Kamper et al., 2006). The genomic organization of a symbiotic fungal species can now be studied with the increase in genome-sequence projects and the availability of the genome sequences of the several fungal species (Sharma, 2016). One of the earlier sequenced basidiomycetous fungi, Laccaria bicolor, has genome of 65 million base pairs, and 20,000 predicted genes, which is relatively larger than other fungi (Martin et al., 2008). Only 70% of the predicted genes have homologues in other fungi, and their size can be partly accounted for by a large number of transposons and repeated sequences and by the presence of large lineage-specific multigene families. An earlier report provides the evidences for the expansion of numerous protein gene families related to the functions that make possible the symbiotic relationship between L. bicolor and its host Populus trichocarpa. In contrast, the genome of L. bicolor shows a marked reduction in the gene families coding for plant cell-wall degradation enzymes, while these families are well represented in the genomes of many other fungal pathogens (Martin et al., 2008). Further, in many fungal pathogens, genetic variations created by chromosomal rearrangements have been reported to favor adaptation to novel hosts or nutritional environments (Larriba, 2004). For example, in the pathogenic yeast Candida albicans, phenotypic mutants derived in vitro often exhibit altered karyotypes and mutation frequencies varying between 10− 5 and 10− 2, depending upon the strain (Rustchenko, 2007), whereas, in Fusarium graminearum, the localized and highly polymorphic genomic regions are significantly enriched with genes favoring plant infection, such as secreted proteins, major facilitator transporters, and cytochrome P450s (Cuomo et al., 2007). Phytopathogenic bacteria are a group of bacteria pathogenic to plants and therefore generate large implications on agriculture and food security. Those bacteria are regarded as equally important to agriculture as viral (Scholthof et al., 2011) and fungal pathogens (Dean et al., 2012). Analyses of genome sequences in bacteria have demonstrated that many of the genes required for virulence are restricted to pathogenic organisms and that they have been introduced into the genomes by horizontal gene transfer. Horizontal gene transfer, the nonsexual transfer of genetic material between organisms, is well established as a major evolutionary process in bacteria, for example, bacterial pathogens to acquire new virulence functions (Lovell et  al., 2009). Genes on plasmids or secondary chromosomes have been shown to evolve faster, and thus, together with the capability of exchange, plasmids can represent a hot spot of evolution for phytopathogenic bacteria (Cooper et al., 2010).

16  COMPARATIVE GENOMICS TO STUDY PLANT-PATHOGEN COEVOLUTION The plant-pathogen coevolution has repeatedly implicated the gene-to-gene interaction, as a primary interface at which pathogens prudently evolve to evade their presence and plants evolve to improve detection. While interacting, plants encode resistance (R) products that



16  Comparative Genomics to Study Plant-Pathogen Coevolution

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e­ ither directly or indirectly recognize the action of specific microbial effectors. This recognition requires the presence of a specific R gene and a specific effector and induces localized cell death, which is a hypersensitive response and a systemic defense response (Jones and Dangl, 2006). Recent comparative genomics studies have revealed that the genes involved in plant defense and pathogen virulence are among the most polymorphic in the respective genomes (Karasov et al., 2014). Resistance and virulence genes exhibit exceptional levels of variation, in part due to the genomic processes that generate it and the selection that promotes its maintenance. The persistence of this variation is unexpected on the basis of the dynamics that we see in agriculture, yet in nature, there are clear indications of diversifying and balancing selection in action (Karasov et al., 2014). Comparative genomics of pathogens also shows that the genes encoding effectors are not randomly distributed in the genome. Genomes of many plant pathogens are compartmentalized into gene-dense and gene-poor regions rich in transposable elements. Such genome compartmentalization in plant pathogens has been called the “two-speed genome.” Genes encoding putative effectors were found to be enriched in the gene-poor compartments, which show higher rates of evolution (Plissonneau et  al., 2016). In general, effectors are expected to mutate frequently, enabling pathogens to avoid detection within extant hosts or adapt to new hosts (Ma et al., 2006; Lovell et al., 2011). The chromosomes with effector loci are mostly located in genomic island that can be reshuffled or transferred between pathogens or lost (Ma et al., 2006; McCann and Guttman, 2008). The frequent exchange of genetic material can result in chimeric genomes, as reported in Xanthomonas species, which contain genes from groups as disparate as the Archaea and Eukarya (Lima et al., 2008). In Brassica phoma stem-canker pathogen Leptosphaeria maculans, the genome has experienced a recent and massive invasion of transposable elements that led to two distinct types of isochors (Grandaubert et al., 2014; Rouxel et al., 2011). The GC isochors are gene-dense, while the ATrich isochors only contain 5% of the predicted genes and are mainly composed of a mosaic of transposable elements. They are degenerated by repeat-induced point mutations (RIPs), which are a genomic defense that prevents the spread of transposable elements by mutating copies of identical sequences (Galagan and Selker, 2004). Furthermore, AT-rich isochors are enriched in pathogenicity-related genes and can evolve rapidly due to RIPs (Daverdin et al., 2012; Fudal et al., 2009). Later, the analyses of genome architecture and gene expression of P. infestans revealed novel candidate virulence factors in the secretome (Raffaele et  al., 2010). The massively expanded genomes of P. infestans (late blight pathogen of potato) and its closely related species, that is, P. ipomoeae and P. mirabilis, revealed large numbers of gene-poor and repeat-rich compartments that are enriched in effector genes (Raffaele et al., 2010). Recently, a complete assembly of the genome of the highly virulent and polymorphic fungal wheat pathogen, Z. tritici isolate 3D7, has been reported (Plissonneau et al., 2016). With the combination of single-molecule real-time sequencing, genetic maps, and transcriptomics data, a fully assembled and annotated genome of the highly virulent field isolate 3D7 was generated. Comparative genomics analyses against the complete reference genome IPO323 identified large chromosomal inversions and the complete gain or loss of transposable-element clusters, explaining the extensive chromosomal-length polymorphisms found in Z. tritici. Moreover, the orphan genes found in the genomic studies were enriched in genes encoding putative effectors and included a gene that is one of the most upregulated putative effector genes during wheat infection (Plissonneau et al., 2016).

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In plants, R genes are the most polymorphic loci in plant genomes, largely because they occur in clusters of paralogous copies (Michelmore and Meyers, 1998). Their high sequence similarity and repetitive sequences (e.g., the leucine-rich repeat (LRR) domain common to most R genes) predispose them to slippage and nonallelic homologous recombination and lead to their frequent turnover and diversification (fusion, duplication, or deletion) (Nagy and Bennetzen, 2008; Wicker et  al., 2007). Allelic variation in many R genes is likely to be ecologically and functionally relevant. Population genetic analyses in several plant species provide insight into the adaptive significance of this variation, identifying dozens of R genes likely to have undergone diversifying selection (Karasov et al., 2014). For example, elevated Ka/Ks ratios are common in the LRRs of paralogous R genes and suggest the rapid diversification of LRRs within a species (Karasov et al., 2014).

17  METAGENOMIC ANALYSIS: METADATA OF OBLIGATE BIOTROPHS The patterns are becoming more apparent relating genomes to lifestyles; therefore, there is a need to recover further genomes of obligate biotrophs, including isolates from wild plants. These data should also be connected to studies generating metadata, including information on pathogen lifestyles, host characteristics, pathogen genomes (i.e., on haplont and dikaryon host), and metagenomes collected from the associated microbiome (Kemen et al., 2015). In metagenomic studies of plant microbiota, it remains challenging to obtain a comprehensive representation of all constituent microorganisms. Currently, the complexity of rhizosphere and soil communities prevents the de novo assembly of a sizable fraction of metagenomes, in particular for low-abundance community members. Thus, for their study and draft genome reconstruction for community members, sequencing of reference genomes from isolated cultures, as in human microbiome studies, or by single-cell sequencing is likely to be helpful. Longer read lengths would further improve assembly, data binning, and draft genome reconstruction from metagenomes. Single-molecule sequencing technologies such as PacBio SMRT technology now deliver longer reads, which could enable substantial advances in the study of complex microbial communities (Guttman et al., 2014). The set of features required for bacteria to efficiently colonize the rhizosphere is yet to be properly described. For instance, studies have shown the importance of motility (de Weger et al., 1987) and lipopolysaccharide (LPS) production (de Weger et al., 1989) for the colonization of potato roots by P. fluorescens. The capacity to form biofilm was shown to be related to the rhizosphere colonization in Bacillus amyloliquefaciens and rhizobia species (Rinaudi and Giordano, 2010; Tan et al., 2013), but not in P. fluorescens (Barahona et al., 2010). Fast growth rate was suggested to be important for rhizosphere colonization in Pseudomonas spp. and B. amyloliquefaciens (Tan et al., 2013). There are increasing indications that the rhizosphere could be a hot spot of HGT events, for example, by the increase of the transference of conjugative plasmids between rhizosphere inhabitants (Pukall et  al., 1996; Van Elsas et  al., 1998). The suitability of bacteria for HGT processes could hypothetically support the rapid adaptation of bacteria in the face of environmental shifts. Recently, key bacterial traits for rhizosphere colonization using sugarcane as a model system have been identified by Lopes et al. (2016). The bacterial communities and assessed



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shifts in the taxonomic and functional profiles in bulk soil in comparison with the rhizosphere have been analyzed using a combination of bacterial quantification, metabolic capacity to degrade carbon sources, high-throughput sequencing, and metagenome prediction. Metagenomic approach was used to better understand about the taxonomic and ecological relationships that microorganisms establish in the sugarcane rhizosphere and some of the characteristics needed for bacterial communities to colonize this soil habitat, including polygalacturonase activity and the possible importance of HGT in the rhizosphere (Lopes et al., 2016). Earlier, the bulk soil community has shown a higher ratio of functions related to secondary metabolism, including the degradation of complex compounds and environmental adaptation. A deeper investigation of the metagenome prediction showed a greater abundance of genes related to colanic acid biosynthesis protein (WcaH) in the rhizosphere. Colanic acid is associated with biofilm formation in Escherichia coli (Rinaudi and Giordano, 2010; Tan et al., 2013). Another interesting result obtained from the recent metagenome prediction was the higher abundance of genes related to HGT in the rhizosphere and genes related to bacterial transformation and conjugation, such as those associated with the type IV secretion system, enriched in the rhizosphere (Lopes et al., 2016).

18  CONCLUSION AND FUTURE PROSPECTS The generation of huge amount of sequencing data for the characterization of complex microbial communities has resulted in the development of microbiome research. The recent technical advancement has provided an in-depth description of microbial phylogeny and plant-microbe interactions. These developments helped in understanding the mechanisms of plant-microbe interactions for their adaptations under stress conditions. Their interactions help in the production of valuable metabolites with diverse biological roles. Microbiome research will help in understanding the taxonomical, environmental, agricultural, and biomedical aspects of the field. The plant-microbe interaction will also reveal the functioning of gene expression, metabolic pathways, protein levels, subcellular localization required for their growth and development. Furthermore, the role of recent technology will help in the development of sustainable crop production to feed the increasing world population.

Acknowledgment The authors acknowledge the financial assistance by the Department of Biotechnology, Government of India (DBTIPLS grant no. BT/PR13563/MED/12/425/2010).

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Further Reading Halmschlager, E., Butin, H., Donaubauer, E., 1993. Endophytic fungi in leaves and twigs of Quercus petraea. Eur. J. For. Pathol. 23, 51–63. Meyers, B.C., Shen, K.A., Rohani, P., Gaut, B.S., Michelmore, R.W., 1998. Receptor-like genes in the major resistance locus of lettuce are subject to divergent selection. Plant Cell 10, 1833–1846.