Laser Capture Microdissection Transcriptome Reveals Spatiotemporal Tissue Gene Expression Patterns of Medicago truncatula Roots Responding to Rhizobia.

We report a public resource for examining the spatiotemporal RNA expression of 54,893 Medicago truncatula genes during the first 72 h of response to rhizobial inoculation. Using a methodology that allows synchronous inoculation and growth of more than 100 plants in a single media container, we harvested the same segment of each root responding to rhizobia in the initial inoculation over a time course, collected individual tissues from these segments with laser capture microdissection, and created and sequenced RNA libraries generated from these tissues. We demonstrate the utility of the resource by examining the expression patterns of a set of genes induced very early in nodule signaling, as well as two gene families (CLE peptides and nodule specific PLAT-domain proteins) and show that despite similar whole-root expression patterns, there are tissue differences in expression between the genes. Using a rhizobial response dataset generated from transcriptomics on intact root segments, we also examined differential temporal expression patterns and determined that, after nodule tissue, the epidermis and cortical cells contained the most temporally patterned genes. We circumscribed gene lists for each time and tissue examined and developed an expression pattern visualization tool. Finally, we explored transcriptomic differences between the inner cortical cells that become nodules and those that do not, confirming that the expression of 1-aminocyclopropane-1-carboxylate synthases distinguishes inner cortical cells that become nodules and provide and describe potential downstream genes involved in early nodule cell division. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Mutation of BAM2 rescues the sunn hypernodulation phenotype in Medicago truncatula, suggesting that a signaling pathway like CLV1/BAM in Arabidopsis affects nodule number.

The unique evolutionary adaptation of legumes for nitrogen-fixing symbiosis leading to nodulation is tightly regulated by the host plant. The autoregulation of nodulation (AON) pathway negatively regulates the number of nodules formed in response to the carbon/nitrogen metabolic status of the shoot and root by long-distance signaling to and from the shoot and root. Central to AON signaling in the shoots of Medicago truncatula is SUNN, a leucine-rich repeat receptor-like kinase with high sequence similarity with CLAVATA1 (CLV1), part of a class of receptors in Arabidopsis involved in regulating stem cell populations in the root and shoot. This class of receptors in Arabidopsis includes the BARELY ANY MERISTEM family, which, like CLV1, binds to CLE peptides and interacts with CLV1 to regulate meristem development. M. truncatula contains five members of the BAM family, but only MtBAM1 and MtBAM2 are highly expressed in the nodules 48 hours after inoculation. Plants carry mutations in individual MtBAMs, and several double BAM mutant combinations all displayed wild-type nodule number phenotypes. However, Mtbam2 suppressed the sunn-5 hypernodulation phenotype and partially rescued the short root length phenotype of sunn-5 when present in a sunn-5 background. Grafting determined that bam2 suppresses supernodulation from the roots, regardless of the SUNN status of the root. Overexpression of MtBAM2 in wild-type plants increases nodule numbers, while overexpression of MtBAM2 in some sunn mutants rescues the hypernodulation phenotype, but not the hypernodulation phenotypes of AON mutant rdn1-2 or crn. Relative expression measurements of the nodule transcription factor MtWOX5 downstream of the putative bam2 sunn-5 complex revealed disruption of meristem signaling; while both bam2 and bam2 sunn-5 influence MtWOX5 expression, the expression changes are in different directions. We propose a genetic model wherein the specific root interactions of BAM2/SUNN are critical for signaling in nodule meristem cell homeostasis in M. truncatula.

Common gene expression patterns are observed in rice roots during associations with plant growth-promoting bacteria, Herbaspirillum seropedicae and Azospirillum brasilense.

Non-legume plants such as rice and maize can form beneficial associations with plant growth-promoting bacteria (PGPB) such as Herbaspirillum seropedicae and Azospirillum brasilense. Several studies have shown that these PGPB promote plant growth via multiple mechanisms. Our current understanding of the molecular aspects and signaling between plants like rice and PGPB like Herbaspirillum seropedicae is limited. In this study, we used an experimental system where H. seropedicae could colonize the plant roots and promote growth in wild-type rice. Using this experimental setup, we identified 1688 differentially expressed genes (DEGs) in rice roots, 1 day post-inoculation (dpi) with H. seropedicae. Several of these DEGs encode proteins involved in the flavonoid biosynthetic pathway, defense, hormone signaling pathways, and nitrate and sugar transport. We validated the expression pattern of some genes via RT-PCR. Next, we compared the DEGs identified in this study to those we previously identified in rice roots during associations with another PGPB, Azospirillum brasilense. We identified 628 genes that were differentially expressed during both associations. The expression pattern of these genes suggests that some of these are likely to play a significant role(s) during associations with both H. seropedicae and A. brasilense and are excellent targets for future studies.

Time-course RNA-seq analysis provides an improved understanding of gene regulation during the formation of nodule-like structures in rice.

KEY MESSAGE: Using a time-course RNA-seq analysis we identified transcriptomic changes during formation of nodule-like structures (NLS) in rice and compared rice RNA-seq dataset with a nodule transcriptome dataset in Medicago truncatula. Plant hormones can induce the formation of nodule-like structures (NLS) in plant roots even in the absence of bacteria. These structures can be induced in roots of both legumes and non-legumes. Moreover, nitrogen-fixing bacteria can recognize and colonize these root structures. Therefore, identifying the genetic switches controlling the NLS organogenesis program in crops, especially cereals, can have important agricultural implications. Our recent study evaluated the transcriptomic response occurring in rice roots during NLS formation, 7 days post-treatment (dpt) with auxin, 2,4-D. In this current study, we investigated the regulation of gene expression occurring in rice roots at different stages of NLS formation: early (1-dpt) and late (14-dpt). At 1-dpt and 14-dpt, we identified 1662 and 1986 differentially expressed genes (DEGs), respectively. Gene ontology enrichment analysis revealed that the dataset was enriched with genes involved in auxin response and signaling; and in anatomical structure development and morphogenesis. Next, we compared the gene expression profiles across the three time points (1-, 7-, and 14-dpt) and identified genes that were uniquely or commonly differentially expressed at all three time points. We compared our rice RNA-seq dataset with a nodule transcriptome dataset in Medicago truncatula. This analysis revealed there is some amount of overlap between the molecular mechanisms governing nodulation and NLS formation. We also identified that some key nodulation genes were not expressed in rice roots during NLS formation. We validated the expression pattern of several genes via reverse transcriptase polymerase chain reaction (RT-PCR). The DEGs identified in this dataset may serve as a useful resource for future studies to characterize the genetic pathways controlling NLS formation in cereals.

RNA-seq reveals differentially expressed genes in rice (Oryza sativa) roots during interactions with plant-growth promoting bacteria, Azospirillum brasilense.

Major non-legume crops can form beneficial associations with nitrogen-fixing bacteria like Azospirillum brasilense. Our current understanding of the molecular aspects and signaling that occur between important crops like rice and these nitrogen-fixing bacteria is limited. In this study, we used an experimental system where the bacteria could colonize the plant roots and promote plant growth in wild type rice and symbiotic mutants (dmi3 and pollux) in rice. Our data suggest that plant growth promotion and root penetration is not dependent on these genes. We then used this colonization model to identify regulation of gene expression at two different time points during this interaction: at 1day post inoculation (dpi), we identified 1622 differentially expressed genes (DEGs) in rice roots, and at 14dpi, we identified 1995 DEGs. We performed a comprehensive data mining to classify the DEGs into the categories of transcription factors (TFs), protein kinases (PKs), and transporters (TRs). Several of these DEGs encode proteins that are involved in the flavonoid biosynthetic pathway, defense, and hormone signaling pathways. We identified genes that are involved in nitrate and sugar transport and are also implicated to play a role in other plant-microbe interactions. Overall, findings from this study will serve as an excellent resource to characterize the host genetic pathway controlling the interactions between non-legumes and beneficial bacteria which can have long-term implications towards sustainably improving agriculture.

Comparative transcriptome analysis provides key insights into gene expression pattern during the formation of nodule-like structures in Brachypodium.

Auxins can induce the formation of nodule-like structures (NLS) in plant roots even in the absence of rhizobia and nitrogen-fixing bacteria can colonize these structures. Interestingly, NLS can be induced in roots of both legumes and non-legumes. However, our understanding of NLS formation in non-legumes at a molecular level is limited. This study aims to investigate NLS formation at a developmental and molecular level in Brachypodium distachyon. We treated Brachypodium roots with the synthetic auxin, 2,4-D, to induce NLS at a high frequency (> 80%) under controlled conditions. A broad base and a diffuse meristem characterized these structures. Next, we performed a comprehensive RNA-sequencing experiment to identify differentially expressed genes (DEGs) in Brachypodium roots during NLS formation. We identified 618 DEGs; several of which are promising candidates for control of NLS based on their biological and molecular functions. We validated the expression pattern of several genes via RT-PCR. Next, we compared the expression profile of Brachypodium roots with rice roots during NLS formation. We identified 76 single-copy ortholog pairs in rice and Brachypodium that are both differentially expressed during this process. Some of these genes are involved in auxin signaling, root development, and legume-rhizobia symbiosis. We established an experimental system to study NLS formation in Brachypodium at a developmental and genetic level, and used RNA-sequencing analysis to understand the molecular mechanisms controlling this root organogenesis program. Furthermore, our comparative transcriptome analysis in Brachypodium and rice identified a key set of genes for further investigating this genetic pathway in grasses.

A Developmental and Molecular View of Formation of Auxin-Induced Nodule-Like Structures in Land Plants.

Several studies have shown that plant hormones play important roles during legume-rhizobia symbiosis. For instance, auxins induce the formation of nodule-like structures (NLSs) on legume roots in the absence of rhizobia. Furthermore, these NLS can be colonized by nitrogen-fixing bacteria, which favor nitrogen fixation compared to regular roots and subsequently increase plant yield. Interestingly, auxin also induces similar NLS in cereal roots. While several genetic studies have identified plant genes controlling NLS formation in legumes, no studies have investigated the genes involved in NLS formation in cereals. In this study, first we established an efficient experimental system to induce NLS in rice roots, using auxin, 2,4-D, consistently at a high frequency (>90%). We were able to induce NLS at a high frequency in Medicago truncatula under similar conditions. NLS were characterized by a broad base, a diffuse meristem, and increased cell differentiation in the vasculature. Interestingly, NLS formation appeared very similar in both rice and Medicago, suggesting a similar developmental program. We show that NLS formation in both rice and Medicago occurs downstream of the common symbiotic pathway. Furthermore, NLS formation occurs downstream of cytokinin-induced step(s). We performed a comprehensive RNA sequencing experiment to identify genes differentially expressed during NLS formation in rice and identified several promising genes for control of NLS based on their biological and molecular functions. We validated the expression patterns of several genes using reverse transcription polymerase chain reaction and show varied expression patterns of these genes during different stages of NLS formation. Finally, we show that NLS induced on rice roots under these conditions can be colonized by nitrogen-fixing bacteria, Azorhizobium caulinodans.