Recent advances in understanding the molecular basis of infantile haemangioma development.

Infantile haemangioma (IH) - the most common vascular tumour of infancy - is comprised of diverse cell types, including endothelial cells, pericytes, fibroblasts and immune cells. IH is characterized by rapid proliferation followed by slow involution over 1-10 years. Most lesions regress spontaneously, but up to 10% can be disfiguring, with complications that require further medical treatment. Recent research has revealed the biological characteristics of IH, highlighting the involvement of angiogenesis and vasculogenesis during tumour formation. Gene expression profiling has provided vital insights into the underlying biological processes, with some of the key IH-related pathways identified, including vascular endothelial growth factor, the renin-angiotensin-aldosterone system, hypoxia-inducible factor 1α, Notch, platelet-derived growth factor, phosphoinositide 3-kinase/Akt/mammalian target of rapamycin, Janus kinase/signal transducers and activators of transcription, fibroblast growth factor, peroxisome proliferator-activated receptor-γ and insulin-like growth factor. Further evidence suggests extracellular matrix factors and hormone receptors regulate IH progression. In this review, we explore the molecular mechanisms involved in the proliferating, plateau and involuting phases of IH, identifying differentially expressed genes, targeted proteins and key signalling pathways. This knowledge will increase the broader understanding of vascular development, tissue remodelling and angiogenesis.

Infantile haemangioma is a common vascular tumour (or birthmark) that affects babies. Less is known about the pathways involved in their development and progression. We look at the key proteins involved in the development of these tumours. These proteins help new blood vessels grow (called 'angiogenesis') when a tumour is just starting to appear. The area around the tumour becomes inflamed, and the immune system releases molecules called 'cytokines' that help new blood vessels grow and to increase the number of cells (called 'proliferation'). Within a year, the tumours shrink and are gradually replaced by fatty tissue (called 'spontaneous involution'). This phase is influenced by other proteins called 'IGF' and 'PPAR-γ', which encourage cell death and tissue remodelling. Hormone receptors and the area outside cells called the 'extracellular matrix' could be the key to understanding this change. Even though many protein signalling pathways are involved in the two phases, we still do not know exactly how the switch between them happens. This review explains the molecular mechanisms behind infantile haemangiomas, focusing on how the protein signalling pathways work together. Our research offers valuable insights for both researchers and clinicians in this field.

Tomato deploys defence and growth simultaneously to resist bacterial wilt disease.

Plant disease limits crop production, and host genetic resistance is a major means of control. Plant pathogenic Ralstonia causes bacterial wilt disease and is best controlled with resistant varieties. Tomato wilt resistance is multigenic, yet the mechanisms of resistance remain largely unknown. We combined metaRNAseq analysis and functional experiments to identify core Ralstonia-responsive genes and the corresponding biological mechanisms in wilt-resistant and wilt-susceptible tomatoes. While trade-offs between growth and defence are common in plants, wilt-resistant plants activated both defence responses and growth processes. Measurements of innate immunity and growth, including reactive oxygen species production and root system growth, respectively, validated that resistant plants executed defence-related processes at the same time they increased root growth. In contrast, in wilt-susceptible plants roots senesced and root surface area declined following Ralstonia inoculation. Wilt-resistant plants repressed genes predicted to negatively regulate water stress tolerance, while susceptible plants repressed genes predicted to promote water stress tolerance. Our results suggest that wilt-resistant plants can simultaneously promote growth and defence by investing in resources that act in both processes. Infected susceptible plants activate defences, but fail to grow and so succumb to Ralstonia, likely because they cannot tolerate the water stress induced by vascular wilt.

Ralstonia solanacearum Effectors Localize to Diverse Organelles in Solanum Hosts.

Phytopathogenic bacteria secrete type III effector (T3E) proteins directly into host plant cells. T3Es can interact with plant proteins and frequently manipulate plant host physiological or developmental processes. The proper subcellular localization of T3Es is critical for their ability to interact with plant targets, and knowledge of T3E localization can be informative for studies of effector function. Here we investigated the subcellular localization of 19 T3Es from the phytopathogenic bacteria Ralstonia pseudosolanacearum and Ralstonia solanacearum. Approximately 45% of effectors in our library localize to both the plant cell periphery and the nucleus, 15% exclusively to the cell periphery, 15% exclusively to the nucleus, and 25% to other organelles, including tonoplasts and peroxisomes. Using tomato hairy roots, we show that T3E localization is similar in both leaves and roots and is not impacted by Solanum species. We find that in silico prediction programs are frequently inaccurate, highlighting the value of in planta localization experiments. Our data suggest that Ralstonia targets a wide diversity of cellular organelles and provides a foundation for developing testable hypotheses about Ralstonia effector function.

What are the Top 10 Unanswered Questions in Molecular Plant-Microbe Interactions?

This article is part of the Top 10 Unanswered Questions in MPMI invited review series.The past few decades have seen major discoveries in the field of molecular plant-microbe interactions. As the result of technological and intellectual advances, we are now able to answer questions at a level of mechanistic detail that we could not have imagined possible 20 years ago. The MPMI Editorial Board felt it was time to take stock and reassess. What big questions remain unanswered? We knew that to identify the fundamental, overarching questions that drive our research, we needed to do this as a community. To reach a diverse audience of people with different backgrounds and perspectives, working in different areas of plant-microbe interactions, we queried the more than 1,400 participants at the 2019 International Congress on Molecular Plant-Microbe Interactions meeting in Glasgow. This group effort resulted in a list of ten, broad-reaching, fundamental questions that influence and inform our research. Here, we introduce these Top 10 unanswered questions, giving context and a brief description of the issues. Each of these questions will be the subject of a detailed review in the coming months. We hope that this process of reflecting on what is known and unknown and identifying the themes that underlie our research will provide a framework to use going forward, giving newcomers a sense of the mystery of the big questions and inspiring new avenues and novel insights.[Formula: see text] Copyright © 2020 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Metabolomics of tomato xylem sap during bacterial wilt reveals Ralstonia solanacearum produces abundant putrescine, a metabolite that accelerates wilt disease.

Ralstonia solanacearum thrives in plant xylem vessels and causes bacterial wilt disease despite the low nutrient content of xylem sap. We found that R. solanacearum manipulates its host to increase nutrients in tomato xylem sap, enabling it to grow better in sap from infected plants than in sap from healthy plants. Untargeted GC/MS metabolomics identified 22 metabolites enriched in R. solanacearum-infected sap. Eight of these could serve as sole carbon or nitrogen sources for R. solanacearum. Putrescine, a polyamine that is not a sole carbon or nitrogen source for R. solanacearum, was enriched 76-fold to 37 µM in R. solanacearum-infected sap. R. solanacearum synthesized putrescine via a SpeC ornithine decarboxylase. A ΔspeC mutant required ≥ 15 µM exogenous putrescine to grow and could not grow alone in xylem even when plants were treated with putrescine. However, co-inoculation with wildtype rescued ΔspeC growth, indicating R. solanacearum produced and exported putrescine to xylem sap. Intriguingly, treating plants with putrescine before inoculation accelerated wilt symptom development and R. solanacearum growth and systemic spread. Xylem putrescine concentration was unchanged in putrescine-treated plants, so the exogenous putrescine likely accelerated disease indirectly by affecting host physiology. These results indicate that putrescine is a pathogen-produced virulence metabolite.

Plant pathogens and symbionts target the plant nucleus.

In plant-microbe interactions, symbionts and pathogens live within plants and attempt to avoid triggering plant defense responses. In order to do so, these microbes have evolved multiple mechanisms that target components of the plant cell nucleus. Rhizobia-induced symbiotic signaling requires the function of specific legume nucleoporins within the nuclear pore complex. Symbiont and pathogen effectors harbor nuclear localization sequences that facilitate movement across nuclear pores, allowing these proteins to target transcription factors that function in defense. Oomycete pathogens introduce proteins that interact with plant pre-mRNA splicing components in order to alter host splicing of defense-related transcripts. Together, these functions indicate that the nucleus is an active site of symbiotic and pathogenic functioning in plant-microbe interactions.

EffectorK, a comprehensive resource to mine for Ralstonia, Xanthomonas, and other published effector interactors in the Arabidopsis proteome.

Pathogens deploy effector proteins that interact with host proteins to manipulate the host physiology to the pathogen's own benefit. However, effectors can also be recognized by host immune proteins, leading to the activation of defence responses. Effectors are thus essential components in determining the outcome of plant-pathogen interactions. Despite major efforts to decipher effector functions, our current knowledge on effector biology is scattered and often limited. In this study, we conducted two systematic large-scale yeast two-hybrid screenings to detect interactions between Arabidopsis thaliana proteins and effectors from two vascular bacterial pathogens: Ralstonia pseudosolanacearum and Xanthomonas campestris. We then constructed an interactomic network focused on Arabidopsis and effector proteins from a wide variety of bacterial, oomycete, fungal, and invertebrate pathogens. This network contains our experimental data and protein-protein interactions from 2,035 peer-reviewed publications (48,200 Arabidopsis-Arabidopsis and 1,300 Arabidopsis-effector protein interactions). Our results show that effectors from different species interact with both common and specific Arabidopsis interactors, suggesting dual roles as modulators of generic and adaptive host processes. Network analyses revealed that effector interactors, particularly "effector hubs" and bacterial core effector interactors, occupy important positions for network organization, as shown by their larger number of protein interactions and centrality. These interactomic data were incorporated in EffectorK, a new graph-oriented knowledge database that allows users to navigate the network, search for homology, or find possible paths between host and/or effector proteins. EffectorK is available at www.effectork.org and allows users to submit their own interactomic data.

The genetic network controlling the Arabidopsis transcriptional response to Pseudomonas syringae pv. maculicola: roles of major regulators and the phytotoxin coronatine.

Expression profiling of wild-type plants and mutants with defects in key components of the defense signaling network was used to model the Arabidopsis network 24 h after infection by Pseudomonas syringae pv. maculicola ES4326. Results using the Affymetrix ATH1 array revealed that expression levels of most pathogen-responsive genes were affected by mutations in coi1, ein2, npr1, pad4, or sid2. These five mutations defined a small number of different expression patterns displayed by the majority of pathogen-responsive genes. P. syringae pv. tomato strain DC3000 elicited a much weaker salicylic acid (SA) response than ES4326. Additional mutants were profiled using a custom array. Profiles of pbs3 and ndr1 revealed major effects of these mutations and allowed PBS3 and NDR1 to be placed between the EDS1/PAD4 node and the SA synthesis node in the defense network. Comparison of coi1, dde2, and jar1 profiles showed that many genes were affected by coi1 but very few were affected by dde2 or jar1. Profiles of coi1 plants infected with ES4326 were very similar to those of wild-type plants infected with bacteria unable to produce the phytotoxin coronatine, indicating that, essentially, all COI1-dependent gene expression changes in this system are caused by coronatine.

Ralstonia solanacearum requires PopS, an ancient AvrE-family effector, for virulence and To overcome salicylic acid-mediated defenses during tomato pathogenesis.

UNLABELLED: During bacterial wilt of tomato, the plant pathogen Ralstonia solanacearum upregulates expression of popS, which encodes a type III-secreted effector in the AvrE family. PopS is a core effector present in all sequenced strains in the R. solanacearum species complex. The phylogeny of popS mirrors that of the species complex as a whole, suggesting that this is an ancient, vertically inherited effector needed for association with plants. A popS mutant of R. solanacearum UW551 had reduced virulence on agriculturally important Solanum spp., including potato and tomato plants. However, the popS mutant had wild-type virulence on a weed host, Solanum dulcamara, suggesting that some species can avoid the effects of PopS. The popS mutant was also significantly delayed in colonization of tomato stems compared to the wild type. Some AvrE-type effectors from gammaproteobacteria suppress salicylic acid (SA)-mediated plant defenses, suggesting that PopS, a betaproteobacterial ortholog, has a similar function. Indeed, the popS mutant induced significantly higher expression of tomato SA-triggered pathogenesis-related (PR) genes than the wild type. Further, pretreatment of roots with SA exacerbated the popS mutant virulence defect. Finally, the popS mutant had no colonization defect on SA-deficient NahG transgenic tomato plants. Together, these results indicate that this conserved effector suppresses SA-mediated defenses in tomato roots and stems, which are R. solanacearum's natural infection sites. Interestingly, PopS did not trigger necrosis when heterologously expressed in Nicotiana leaf tissue, unlike the AvrE homolog DspEPcc from the necrotroph Pectobacterium carotovorum subsp. carotovorum. This is consistent with the differing pathogenesis modes of necrosis-causing gammaproteobacteria and biotrophic R. solanacearum. IMPORTANCE: The type III-secreted AvrE effector family is widely distributed in high-impact plant-pathogenic bacteria and is known to suppress plant defenses for virulence. We characterized the biology of PopS, the only AvrE homolog made by the bacterial wilt pathogen Ralstonia solanacearum. To our knowledge, this is the first study of R. solanacearum effector function in roots and stems, the natural infection sites of this pathogen. Unlike the functionally redundant R. solanacearum effectors studied to date, PopS is required for full virulence and wild-type colonization of two natural crop hosts. R. solanacearum is a biotrophic pathogen that causes a nonnecrotic wilt. Consistent with this, PopS suppressed plant defenses but did not elicit cell death, unlike AvrE homologs from necrosis-causing plant pathogens. We propose that AvrE family effectors have functionally diverged to adapt to the necrotic or nonnecrotic lifestyle of their respective pathogens.

A high-performance, small-scale microarray for expression profiling of many samples in Arabidopsis-pathogen studies.

Studies of the behavior of biological systems often require monitoring of the expression of many genes in a large number of samples. While whole-genome arrays provide high-quality gene-expression profiles, their high cost generally limits the number of samples that can be studied. Although inexpensive small-scale arrays representing genes of interest could be used for many applications, it is challenging to obtain accurate measurements with conventional small-scale microarrays. We have developed a small-scale microarray system that yields highly accurate and reproducible expression measurements. This was achieved by implementing a stable gene-based quantile normalization method for array-to-array normalization, and a probe-printing design that allows use of a statistical model to correct for effects of print tips and uneven hybridization. The array measures expression values in a single sample, rather than ratios between two samples. This allows accurate comparisons among many samples. The array typically yielded correlation coefficients higher than 0.99 between technically duplicated samples. Accuracy was demonstrated by a correlation coefficient of 0.88 between expression ratios determined from this array and an Affymetrix GeneChip, by quantitative RT-PCR, and by spiking known amounts of specific RNAs into the RNA samples used for profiling. The array was used to compare the responses of wild-type, rps2 and ndr1 mutant plants to infection by a Pseudomonas syringae strain expressing avrRpt2. The results suggest that ndr1 affects a defense-signaling pathway(s) in addition to the RPS2-dependent pathway, and indicate that the microarray is a powerful tool for systems analyses of the Arabidopsis disease-signaling network.

Medicago truncatula NIN is essential for rhizobial-independent nodule organogenesis induced by autoactive calcium/calmodulin-dependent protein kinase.

The symbiotic association between legumes and nitrogen-fixing bacteria collectively known as rhizobia results in the formation of a unique plant root organ called the nodule. This process is initiated following the perception of rhizobial nodulation factors by the host plant. Nod factor (NF)-stimulated plant responses, including nodulation-specific gene expression, is mediated by the NF signaling pathway. Plant mutants in this pathway are unable to nodulate. We describe here the cloning and characterization of two mutant alleles of the Medicago truncatula ortholog of the Lotus japonicus and pea (Pisum sativum) NIN gene. The Mtnin mutants undergo excessive root hair curling but are impaired in infection and fail to form nodules following inoculation with Sinorhizobium meliloti. Our investigation of early NF-induced gene expression using the reporter fusion ENOD11::GUS in the Mtnin-1 mutant demonstrates that MtNIN is not essential for early NF signaling but may negatively regulate the spatial pattern of ENOD11 expression. It was recently shown that an autoactive form of a nodulation-specific calcium/calmodulin-dependent protein kinase is sufficient to induce nodule organogenesis in the absence of rhizobia. We show here that MtNIN is essential for autoactive calcium/calmodulin-dependent protein kinase-induced nodule organogenesis. The non-nodulating hcl mutant has a similar phenotype to Mtnin, but we demonstrate that HCL is not required in this process. Based on our data, we suggest that MtNIN functions downstream of the early NF signaling pathway to coordinate and regulate the correct temporal and spatial formation of root nodules.

An ERF transcription factor in Medicago truncatula that is essential for Nod factor signal transduction.

Rhizobial bacteria activate the formation of nodules on the appropriate host legume plant, and this requires the bacterial signaling molecule Nod factor. Perception of Nod factor in the plant leads to the activation of a number of rhizobial-induced genes. Putative transcriptional regulators in the GRAS family are known to function in Nod factor signaling, but these proteins have not been shown to be capable of direct DNA binding. Here, we identify an ERF transcription factor, ERF Required for Nodulation (ERN), which contains a highly conserved AP2 DNA binding domain, that is necessary for nodulation. Mutations in this gene block the initiation and development of rhizobial invasion structures, termed infection threads, and thus block nodule invasion by the bacteria. We show that ERN is necessary for Nod factor-induced gene expression and for spontaneous nodulation activated by the calcium- and calmodulin-dependent protein kinase, DMI3, which is a component of the Nod factor signaling pathway. We propose that ERN is a component of the Nod factor signal transduction pathway and functions downstream of DMI3 to activate nodulation gene expression.

Nitrogen fixation mutants of Medicago truncatula fail to support plant and bacterial symbiotic gene expression.

The Rhizobium-legume symbiosis culminates in the exchange of nutrients in the root nodule. Bacteria within the nodule reduce molecular nitrogen for plant use and plants provide bacteria with carbon-containing compounds. Following the initial signaling events that lead to plant infection, little is known about the plant requirements for establishment and maintenance of the symbiosis. We screened 44,000 M2 plants from fast neutron-irradiated Medicago truncatula seeds and isolated eight independent mutant lines that are defective in nitrogen fixation. The eight mutants are monogenic and represent seven complementation groups. To monitor bacterial status in mutant nodules, we assayed Sinorhizobium meliloti symbiosis gene promoters (nodF, exoY, bacA, and nifH) in the defective in nitrogen fixation mutants. Additionally, we used an Affymetrix oligonucleotide microarray to monitor gene expression changes in wild-type and three mutant plants during the nodulation process. These analyses suggest the mutants can be separated into three classes: one class that supports little to no nitrogen fixation and minimal bacterial expression of nifH; another class that supports no nitrogen fixation and minimal bacterial expression of nodF, bacA, and nifH; and a final class that supports low levels of both nitrogen fixation and bacterial nifH expression.

Nodulation signaling in legumes requires NSP2, a member of the GRAS family of transcriptional regulators.

Rhizobial bacteria enter a symbiotic interaction with legumes, activating diverse responses in roots through the lipochito oligosaccharide signaling molecule Nod factor. Here, we show that NSP2 from Medicago truncatula encodes a GRAS protein essential for Nod-factor signaling. NSP2 functions downstream of Nod-factor-induced calcium spiking and a calcium/calmodulin-dependent protein kinase. We show that NSP2-GFP expressed from a constitutive promoter is localized to the endoplasmic reticulum/nuclear envelope and relocalizes to the nucleus after Nod-factor elicitation. This work provides evidence that a GRAS protein transduces calcium signals in plants and provides a possible regulator of Nod-factor-inducible gene expression.

Plant and bacterial symbiotic mutants define three transcriptionally distinct stages in the development of the Medicago truncatula/Sinorhizobium meliloti symbiosis.

In the Medicago truncatula/Sinorhizobium meliloti symbiosis, the plant undergoes a series of developmental changes simultaneously, creating a root nodule and allowing bacterial entry and differentiation. Our studies of plant genes reveal novel transcriptional regulation during the establishment of the symbiosis and identify molecular markers that distinguish classes of plant and bacterial symbiotic mutants. We have identified three symbiotically regulated plant genes encoding a beta,1-3 endoglucanase (MtBGLU1), a lectin (MtLEC4), and a cysteine-containing protein (MtN31). MtBGLU1 is down-regulated in the plant 24 h after exposure to the bacterial signal, Nod factor. The non-nodulating plant mutant dmi1 is defective in the ability to down-regulate MtBGLU1. MtLEC4 and MtN31 are induced 1 and 2 weeks after bacterial inoculation, respectively. We examined the regulation of these two genes and three previously identified genes (MtCAM1, ENOD2, and MtLB1) in plant symbiotic mutants and wild-type plants inoculated with bacterial symbiotic mutants. Plant (bit1, rit1, and Mtsym1) and bacterial (exoA and exoH) mutants with defects in the initial stages of invasion are unable to induce MtLEC4, MtN31, MtCAM1, ENOD2, and MtLB1. Bacterial mutants (fixJ and nifD) and a subset of plant mutants (dnf2, dnf3, dnf4, dnf6, and dnf7) defective for nitrogen fixation induce the above genes. The bacA bacterial mutant, which senesces upon deposition into plant cells, and two plant mutants with defects in nitrogen fixation (dnf1 and dnf5) induce MtLEC4 and ENOD2 but not MtN31, MtCAM1, or MtLB1. These data suggest the presence of at least three transcriptionally distinct developmental stages during invasion of M. truncatula by S. meliloti.

Six nonnodulating plant mutants defective for Nod factor-induced transcriptional changes associated with the legume-rhizobia symbiosis.

As the legume-rhizobia symbiosis is established, the plant recognizes bacterial-signaling molecules, Nod factors (NFs), and initiates transcriptional and developmental changes within the root to allow bacterial invasion and the construction of a novel organ, the nodule. Plant mutants defective in nodule initiation (Nod(-)) are thought to have defects in NF-signal transduction. However, it is unknown whether WT plants respond to NF-independent bacterial-derived signals or whether Nod(-) plant mutants show defects in global symbiosis-associated gene expression. To characterize plant gene expression in the establishment of the symbiosis, we used an Affymetrix oligonucleotide microarray representing 9,935 Medicago truncatula expressed sequences. We identified 46 sequences that are differentially expressed in plants exposed for 24 h to WT Sinorhizobium meliloti or to the invasion defective S. meliloti mutant, exoA. Eight of these genes encode nucleolar proteins, which are implicated in ribosome biogenesis. We also identified differentially expressed transcription factors, signaling components, defense response proteins, stress response proteins, and several previously uncharacterized genes. NF appears both necessary and sufficient to induce most changes. Six of seven Nod(-) M. truncatula mutants (nfp, dmi1, dmi2, dmi3, nsp1, and nsp2) showed no transcriptional response to S. meliloti, suggesting that the encoded proteins are required for initiating new transcription. The Nod(-) mutant hcl, however, exhibits a reduced transcriptional response to S. meliloti, indicating that the machinery responsible for initiating new transcription is at least partially functional in this mutant.

Pharmacological analysis of nod factor-induced calcium spiking in Medicago truncatula. Evidence for the requirement of type IIA calcium pumps and phosphoinositide signaling.

Bacterial Nod factors trigger a number of cellular responses in root hairs of compatible legume hosts, which include periodic, transient increases in cytosolic calcium levels, termed calcium spiking. We screened 13 pharmaceutical modulators of eukaryotic signal transduction for effects on Nod factor-induced calcium spiking. The purpose of this screening was 2-fold: to implicate enzymes required for Nod factor-induced calcium spiking in Medicago sp., and to identify inhibitors of calcium spiking suitable for correlating calcium spiking to other Nod factor responses to begin to understand the function of calcium spiking in Nod factor signal transduction. 2-Aminoethoxydiphenylborate, caffeine, cyclopiazonic acid (CPA), 2,5-di-(t-butyl)-1,4-hydroquinone, and U-73122 inhibit Nod factor-induced calcium spiking. CPA and U-73122 are inhibitors of plant type IIA calcium pumps and phospholipase C, respectively, and implicate the requirement for these enzymes in Nod factor-induced calcium spiking. CPA and U-73122 inhibit Nod factor-induced calcium spiking robustly at concentrations with no apparent toxicity to root hairs, making CPA and U-73122 suitable for testing whether calcium spiking is causal to subsequent Nod factor responses.

A Ca2+/calmodulin-dependent protein kinase required for symbiotic nodule development: Gene identification by transcript-based cloning.

In the establishment of the legume-rhizobial symbiosis, bacterial lipochitooligosaccharide signaling molecules termed Nod factors activate the formation of a novel root organ, the nodule. Nod factors elicit several responses in plant root hair cells, including oscillations in cytoplasmic calcium levels (termed calcium spiking) and alterations in root hair growth. A number of plant mutants with defects in the Nod factor signaling pathway have been identified. One such Medicago truncatula mutant, dmi3, exhibits calcium spiking and root hair swelling in response to Nod factor, but fails to initiate symbiotic gene expression or cell divisions for nodule formation. On the basis of these data, it is thought that the dmi3 mutant perceives Nod factor but fails to transduce the signal downstream of calcium spiking. Additionally, the dmi3 mutant is defective in the symbiosis with mycorrhizal fungi, indicating the importance of the encoded protein in multiple symbioses. We report the identification of the DMI3 gene, using a gene cloning method based on transcript abundance. We show that transcript-based cloning is a valid approach for cloning genes in barley, indicating the value of this technology in crop plants. DMI3 encodes a calcium/calmodulin-dependent protein kinase. Mutants in pea sym9 have phenotypes similar to dmi3 and have alterations in this gene. The DMI3 class of proteins is well conserved among plants that interact with mycorrhizal fungi, but it is less conserved in Arabidopsis thaliana, which does not participate in the mycorrhizal symbiosis.