Host genotype-specific rhizosphere fungus enhances drought resistance in wheat

Abstract

Background: The severity and frequency of drought are expected to increase substantially in the coming century and dramatically reduce crop yields. Manipulation of rhizosphere microbiomes is an emerging strategy for mitigating drought stress in agroecosystems. However, little is known about the mechanisms underlying how drought-resistant plant recruitment of specific rhizosphere fungi enhances drought adaptation of drought-sensitive wheats. Here, we investigated microbial community assembly features and functional profiles of rhizosphere microbiomes related to drought-resistant and drought-sensitive wheats by amplicon and shotgun metagenome sequencing techniques. We then established evident linkages between root morphology traits and putative keystone taxa based on microbial inoculation experiments. Furthermore, root RNA sequencing and RT-qPCR were employed to explore the mechanisms how rhizosphere microbes modify plant response traits to drought stresses.

Results: Our results indicated that host plant signature, plant niche compartment, and planting site jointly contribute to the variation of soil microbiome assembly and functional adaptation, with a relatively greater effect of host plant signature observed for the rhizosphere fungi community. Importantly, drought-resistant wheat (Yunhan 618) possessed more diverse bacterial and fungal taxa than that of the drought-sensitive wheat (Chinese Spring), particularly for specific fungal species. In terms of microbial interkingdom association networks, the drought-resistant variety possessed more complex microbial networks. Metagenomics analyses further suggested that the enriched rhizosphere microbiomes belonging to the drought-resistant cultivar had a higher investment in energy metabolism, particularly in carbon cycling, that shaped their distinctive drought tolerance via the mediation of drought-induced feedback functional pathways. Furthermore, we observed that host plant signature drives the differentiation in the ecological role of the cultivable fungal species Mortierella alpine (M. alpina) and Epicoccum nigrum (E. nigrum). The successful colonization of M. alpina on the root surface enhanced the resistance of wheats in response to drought stresses via activation of drought-responsive genes (e.g., CIPK9 and PP2C30). Notably, we found that lateral roots and root hairs were significantly suppressed by co-colonization of a drought-enriched fungus (M. alpina) and a drought-depleted fungus (E. nigrum).

Conclusions: Collectively, our findings revealed host genotypes profoundly influence rhizosphere microbiome assembly and functional adaptation, as well as it provides evidence that drought-resistant plant recruitment of specific rhizosphere fungi enhances drought tolerance of drought-sensitive wheats. These findings significantly underpin our understanding of the complex feedbacks between plants and microbes during drought, and lay a foundation for steering "beneficial keystone biome" to develop more resilient and productive crops under climate change. Video Abstract.

Keywords: Drought; Microbiome; Multi-omics; Rhizosphere; Wheat.

Fig. 1

Experimental flow charts for this study. a Long-term field experiments were established starting in 2007. Two wheat varieties, namely, Chinese Spring (CS) and Yunhan 618 (YH), were employed in this study. Bulk and rhizosphere soils were sampled during the jointing stage for amplicon and metagenomics sequencing. b Procedure for Experiment 1, the rhizosphere soil samples were used to generate microbial suspension cultures to conduct pot experiment. After inoculation, the root samples from different groups were collected for root morphology trait analysis. c Procedure for Experiment 2, isolation of rhizosphere fungi from Experiment 1 and construction of SynComs to confirm the ecological role of specific rhizosphere fungi recruited by plants in enhancing plant drought tolerance. Root-related parameters and plant gene expression were conducted by using phenotype analysis, SEM imaging, and RAN-Seq techniques when the roots were harvested after inoculation

Fig. 2

Patterns of diversity of soil microbiomes and influences of drought stress on enriched bacterial and fungal taxa. a and b represent the Shannon and Chao indices of bacterial and fungal communities, respectively, in all samples. A difference in the letters indicates a significant difference (P < 0.05, n = 5). Abbreviations: Suqian site (SQ), Yangling site (YL), drought-susceptible wheat cultivar Chinese Spring (CS), drought-resistant wheat cultivar Yunhan 618 (YH), bulk soil (B), and rhizosphere soil (T). c Relative abundance of dominant phylum in different wheat cultivars at SQ (left panel) and YL (right panel) sites. d Shared operational taxonomic units (OTUs) in drought-resistant and drought-susceptible cultivars across the YL and SQ sites. e Relative abundances of drought-enriched and drought-depleted OTUs in bacteria and fungi at genus, family, order, and phylum levels

Fig. 3

Microbial interkingdom association networks under drought stress. a Interkingdom co-occurrence networks of drought-resistant and drought-susceptible cultivars at YL and SQ sites. Only compositionality-robust (|ρ|> 0.8) and statistically significant (P < 0.05) correlations are shown. The size of each node indicates the relative abundance of the corresponding amplicon sequence variant (ASV). Blue solid lines represent co-presence associations, and gray dotted lines represent mutual exclusion correlations. The thickness of each line is proportional to the correlation coefficient of the corresponding association. b Bar graph showing the proportion of edges in bacterial–bacterial (BB), bacterial–fungal (BF), and fungal–fungal (FF) correlations in the different samples. c Average numbers of neighbors, edges, and shortest paths in the different samples. d Scatter plot showing the features of degree and closeness centrality for the different wheat cultivars. e Degree and interaction type of the top seven hub nodes in the drought-resistant and drought-susceptible cultivars

Fig. 4

Root morphological traits after different microbial inoculation treatments. Schematic representation of the inoculation experiment. a Five-day-old seedlings were grown in stress-free conditions (CK) or under drought stress (polyethylene glycol [PEG 6000]) and were then either not inoculated (Control) or inoculated with rhizosphere microbiota from the YL or SQ site. b, c The bar graph shows the root length (b) and root diameter (c) of 14-day-old wheat seedlings grown in the control and SQ and YL rhizosphere microbiota groups, respectively. A difference in the lowercase letters indicates a significant difference at a probability level of P < 0.05. d, e Linear regression relationships between the relative abundance of genotype-specific keystone taxa and root length (d) or root diameter (e) after the inoculation treatments. The color of the blue dots indicates the relative abundance of the different species

Fig. 5

Colonization and drought resistance traits after inoculation of roots of wheat plants. a Top: Successful colonization by M. alpina, E. nigrum, and SynComs under drought stress (10% PEG 6000) of roots of CS wheat. Plants not inoculated with fungi served as controls (scale bars: 10 μm). SynCom-2 consisted of M. alpina and E. nigrum (scale bars: 20 μm). SynCom-3 comprised M. alpina, E. nigrum, and SynCom-CS (scale bars: 20 μm). Six biological replicates were measured. Bottom: The effects of SynComs on wheat root tips were recorded using confocal fluorescence microscopy (scale bars: 1 μm). b Growth profiles and root phenotypes of wheat in control group and 18 days after inoculation with SynComs under drought stress. Shoot fresh weights (c) and dry weights (d) of wheat plants in control group and 18 days after inoculation with SynComs under drought stress. A two-sided t-test was used for statistical analyses (n = 3). *P-value < 0.05, **P-value < 0.01

Fig. 6

Synergistic responses between M. alpina and the host resulted in an increase in drought resistance in wheat. a Gene Ontology analysis of differentially expressed genes (DEGs) in cluster 6 in order to identify enriched biological processes, which highlighted a group of abiotic stress response-related and kinase activity that are well known to promote plant responses to drought stress. b Heatmaps constructed using gene expression values (normalized log₂ fold change values) obtained from RNA-Seq data. The gene IDs of DEGs involved in kinase activity and responses to abiotic stress are shown in Table S14. The asterisk displays the difference reached statistical significance (*p < 0.05, **p < 0.01) in M. alpina, E. nigrum, or SynCom-2 in contrast to control. c Relative expression levels (2^−ΔΔCT) obtained by quantitative real-time PCR analysis

Fig. 7

Conceptual paradigm depicting the synergistic mechanisms between host plants and rhizosphere microorganisms that improve drought resistance in wheat. The drought-resistant wheat cultivar determines the direct response of plants to recruit genotype-specific microbial communities, such as M. alpina. M. alpina can feedback to plants and improve drought resistance by activating the CIPK-PP2C network to induce drought-responsive genes. However, M. alpina and E. nigrum together have a negative effect on the lateral roots and root hairs of wheat and lead to wheat becoming significantly more sensitive to drought stress