Employing a combination of high throughput sequencing of microbial marker genes and a bioinformatics pipeline, we characterized the composition of the rice-root associated fungal microbiome under well-watered and drought conditions. The use of an indica rice population consisting of 247 genotyped accessions (Rebolledo et al., 2016; Kadam et al., 2017), allowed us to assess the rice-root mycobiota diversity and to determine the extent to which this diversity is controlled genetically by the host-plant. Drought has a strong impact on the root mycobiota and increases its diversity (Fig. S1b). The same was also recently reported for a tropical grassland soil and in a study on rice (Andreo-Jimenez et al., 2019; Oliveira et al., 2020). Drought caused an increase in species diversity and evenness but at the same time a lower species richness when compared with water-sufficient conditions (Fig. S1b). Despite the large heterogeneity in plant-microbioal communities of a single plant species grown on different soils, a growing body of evidence indicates that the plant microbiota composition is not randomly assembled but ruled by deterministic processes, at least in part, under the control of the host plant (Shi et al., 2018; Xiong et al., 2021). Accordingly, we identified ten SNPs associated with the abundance of specific fungal sequence-clusters and four SNPs linked to sequence-clusters that correlated with yield. The candidate genes underlying these SNPs, encode proteins related to microbial resistance, environmental responses, metabolite transport, and protease and protease inhibitors, which are promising candidates for involvement in the plant-microbiome interaction.
Increasing our knowledge of the plant-microbiome interaction with the final aim to enhance agricultural productivity is a rapidly expanding research field. Little is known about how environmental change such as global warming and drought will affect root based microbial populations, and how, vice versa, these microbial populations contribute to resilience against these environmental challenges. The increase in root-associated fungal diversity under drought potentially equips the host with additional functions to mitigate the consequences of drought (van der Heijden & Hartmann, 2016; Prudent et al., 2020). Under drought, the Pleosporales and Capnodiales, and, to a lesser extent, the Chaetothyriales and Rhizophydiales were more dominant, while the Sordariales and Pezizales were more abundant in the control (Fig. S1a). The Pleosporales are ubiquitous fungi and contain species with quite different functions, such as plant pathogens, saprophytes and plant endophytes, including plant beneficial fungi (Zhang et al., 2009). Inoculation of rice with members of the Pleosporales improved nitrogen content and boosted plant growth (Vergara et al., 2019) and they were reported as key taxa in the rice core seed microbiome (Eyre et al., 2019). Dark Septate Endophytes (DSE), which belong to the Pleosporales, were reported to promote growth of the xerophyte, Ammopiptanthus mongolicus, under drought (Li et al., 2018). A Pleosporales sequence-cluster (OTU_0003) was contributing most to grain yield under well-watered conditions (Fig. S3), making this a highly interesting candidate fungus for follow-up research. The Capnodiales, that were more abundant under drought, are widespread and behave as saprotrophs, plant and human pathogens, mycoparasites and endophytes (Abdollahzadeh et al., 2020). For example, Cladosporium fulvum is a well-studied plant pathogen causing tomato leaf mold (Thomma et al., 2005). A Cladosporium spp. sequence-cluster contributed positively to yield in both treatments (Fig. S1a, S4a). Species such as Cladosporium cladosporioides and Cladosporium porophorum are known fungal endophytes that can hyper-parasitise fungal pathogens of several crops, including rice (Becker et al., 2020; Chaibub et al., 2020; Erfandoust et al., 2020). The Sordariales and Pezizales were more abundant in our control treatment. The former are saprotrophs, and plant pathogenic and wood inhabiting fungi. For instance, Colletotrichum spp. are important plant pathogens that cause anthracnose and rotting diseases and affect a large variety of plant species (Cannon et al., 2012). Pezizales can be saprotrophs, ectomycorrhizae or pathogens in plants. Pezizomycetes, for example, occur in moist habitats in soil and decomposing wood (Ekanayaka et al., 2018). Two sequence-clusters belonging to the Pezizales (Boudiera acanthospora) and Sordariales (Ceratosphaeria lampadophora) positively contributed to yield in the present study (Fig. S3). Ceratosphaeria spp. are closely related to Magnaporthe spp. such as M. grisea, responsible for the rice blast, and occur widely in freshwater environments (Luo et al., 2019), and not much is known about a potential beneficial role.
The positive effect of drought on the microbial diversity has also been reported in artichoke in which the rhizosphere bacterial diversity increased under salt stress (Yang et al., 2016). However, under long-term stress, diversity in ectomycorrhizal fungi in trees decreased again with a few beneficial species remaining as the dominant ones (Gehring et al., 2014). In our study, species richness under drought was lower, just as in coastal plant populations where high salinity resulted in lower AM fungi species richness (Guo & Gong, 2014). The positive correlation of a number of sequence-clusters with plant yield were environment-dependent, with sequence-cluster-yield correlations either in control or drought conditions (Fig. 2a; Fig. S4). This has also been reported for grapevine, in which drought can enhance the growth promoting effect of plant growth promoting bacteria, Acinetobacter and Pseudomonas (Rolli et al., 2015).
A total of 10 different SNPs were found to be associated with the abundance of 6 independent sequence-clusters, from which two also correlated with grain yield under control conditions. We found several candidate genes, putatively involved in plant-microbe interactions, which may be involved in sequence-cluster abundance in rice roots, and possibly in rice yield. When zooming in at the level of individual candidate genes, we found a number of disease resistance and plant development candidate genes. For example, two that encode RALF-LIKE (RALFL) peptides. The RALF protein family is conserved across species and they have a role in root development, and as signaling molecules for stress adaptation responses (e.g. drought) (Sharma et al., 2016). Both RALFL candidate loci are downregulated in rice after root treatment with jasmonic acid (-4 Log2Fold) and abscisic acid (-4 Log2Fold), and Magnaporthe oryzae infection (-3 Log2Fold) (data from RiceXPro database: https://ricexpro.dna.affrc.go.jp/). RALF proteins are able to negatively regulate the plant immune system, and it has been shown that they are differentially expressed upon Botrytis cinerea inoculation in strawberry (Negrini et al., 2020). In Medicago truncatula, RALF genes are induced by nod factors in the early steps of Rhizobium symbiosis (Kereszt et al., 2018). Fungal pathogens like Fusarium oxysporum secrete RALF functional homologues, which was demonstrated to facilitate their infection of roots (Masachis et al., 2016). The RALF genes are of potential interest also because of their role in root development (Sharma et al., 2016). Root developmental genes have been shown to shape the root associated fungal and bacterial microbiome in Arabidopsis (Bergelson et al., 2019), which supports the candidacy of this RALFL gene candidate for sequence-cluster abundance in rice. In our study the two RALFL loci are associated with a sequence-cluster from the Rhizophydiales order which is slightly negatively correlated with yield under drought (Fig. 2). Rhizophydiales are diatom microparasites and are commonly found in water environments, and they compete for microbial photosynthetic carbon from surrounding bacteria, thus affecting the bacterial microbiome composition (Klawonn et al., 2021). We also found several candidate genes related to host-pathogen interactions and defense. One of them, defensin (DEFL), which appears in two loci (DEFL35 LOC_Os01g10550, DELF49 LOC_Os04g31250), is a family of antimicrobial peptides that are involved in defense processes. In rice, two defensin proteins (OsDEF7 and OsDEF8) have been identified to work against two pathogenic bacteria, Xanthomonas oryzae and Erwinia carotovora, and OsAFP1 showed antifungal activity against Candida albicans (Tantong et al., 2016; Ochiai et al., 2018). The defensin proteins found in the present study were associated to a fungus correlating negatively with yield (OTU_0019) and positively/negatively yield-related fungi under drought (DEFL35). This might be an indication that fungal interactions affecting yield more negatively to the rice plant i.e. OTU_0019. On the other hand, DEFL proteins expression pattern is more heterogenous than other pathogen defense related proteins such as NOD-like receptors (NLR), and they present a higher functional diversification i.e. development of reproductive organs, heavy metal resistance (Mondragón-Palomino et al., 2017). It would be interesting to create knock-out mutants of these DEFLs and allelic complementation lines to see what the consequences are for the recruitment of pathogenic and beneficial mycobiota. Another candidate gene, EXOCYST TETHERING COMPLEX (EXO70), is a regulator of secretor-vesicles in the cell membrane, and they are highly specialized. These regulators are involved in several plant development processes, i.e. auxin-dependent root development, pollen maturation, germination, and in immune defense processes (Marković et al., 2021). For instance, EXO70 is involved in the defense against Maganaporthe oryzae in rice (De la Concepcion et al., 2022). On the other hand, these EXO70 tethering complexes are also involved in symbiotic relationships. For instance, EXO70I is required for the development of the periarbuscular membrane during Arbuscular Mycorrhizal (AM) symbiosis in Medicago truncatula (Zhang et al., 2015; Ho-Plágaro et al., 2022). Furthermore, in legumes such as soybean, the tethering system GmExo70J is necessary for the establishment of the symbiosis with nitrogen-fixing rhizobia (Wang et al., 2016). Perhaps our EXO70 candidate gene in rice also plays a role in symbiosis with AM fungi, although, unexpectedly, it is highly expressed in rice leaves (Table 1). Finally, a high number of candidate genes found to be related to OTU_0019 under drought belong to the Bric-a-Brac Tramtrack Broad/Poxvirus and Zinc finger (BTB/POZ) protein complex families that have a role in plant growth and development and plant defense regulation. Interestingly, they can stimulate but also repress the plant immune system (Orosa et al., 2017). For instance, it has been shown in Nicotiana benthamiana that the BTB/POZ complex NbBTB negatively regulates plant resistance against Phytophthora parasitica and it is easily triggered by pathogen effectors (Zhao et al., 2022).
Out of all the fungal-mediated yield SNPs found in our study, there was a clear difference between drought and control, with SNPs on chromosome 1 more present under control and SNPs on chromosome 4 under drought (Fig. 3). These sequence-clusters are interesting molecular targets and the underlying genes could be involved in plant-microbe symbioses as well as in the combined response of the plant-mycobiota and its impact on plant fitness under stressful conditions such as drought, and yield under control conditions. Interestingly among the candidate genes influencing the abundance of yield-related sequence-clusters we found some encoding disease resistance genes (DEFL). As discussed above, receptors related to host defense and symbiosis are structurally similar, making these resistance genes interesting candidates.