A petunia mutant affected in intracellular accommodation and morphogenesis of arbuscular mycorrhizal fungi

The regulation of the arbuscular mycorrhizal (AM) symbiosis is largely under the control of a genetic programme of the plant host. This programme includes a common symbiosis signalling pathway that is shared with the root nodule symbiosis. Whereas this common pathway has been investigated in detail, little is known about the mycorrhiza-specific regulatory steps upstream and downstream of the common pathway. To get further insight in the regulation of the AM symbiosis, a transposon-mutagenized population of Petunia hybrida was screened for mutants with defects in AM development. Here, we describe a petunia mutant, penetration and arbuscule morphogenesis1 (pam1), which is characterized by a strong decrease in colonization by three different AM fungi. Penetrating hyphae are frequently aborted in epidermal cells. Occasionally the fungus can progress to the cortex, but fails to develop arbuscules. The resulting hyphal colonization of the cortex in mutant plants does not support symbiotic acquisition of phosphate and copper by the plant. Expression analysis of three petunia orthologues of the common SYM genes LjPOLLUX, LjSYMRK and MtDMI3 indicates that pam1 is not mutated in these genes. We conclude that the PAM1 gene may play a specific role in intracellular accommodation and morphogenesis of the fungal endosymbiont.     

Medicago truncatula mtpt4 mutants reveal a role for nitrogen in the regulation of arbuscule degeneration in arbuscular mycorrhizal symbiosis

Plants acquire essential mineral nutrients such as phosphorus (P) and nitrogen (N) directly from the soil, but the majority of the vascular plants also gain access to these mineral nutrients through endosymbiotic associations with arbuscular mycorrhizal (AM) fungi. In AM symbiosis, the fungi deliver P and N to the root through branched hyphae called arbuscules. Previously we identified MtPT4, a Medicago truncatula phosphate transporter located in the periarbuscular membrane that is essential for symbiotic phosphate transport and for maintenance of the symbiosis. In mtpt4 mutants arbuscule degeneration occurs prematurely and symbiosis fails. Here, we show that premature arbuscule degeneration occurs in mtpt4 mutants even when the fungus has access to carbon from a nurse plant. Thus, carbon limitation is unlikely to be the primary cause of fungal death. Surprisingly, premature arbuscule degeneration is suppressed if mtpt4 mutants are deprived of nitrogen. In mtpt4 mutants with a low N status, arbuscule lifespan does not differ from that of the wild type, colonization of the mtpt4 root system occurs as in the wild type and the fungus completes its life cycle. Sulphur is another essential macronutrient delivered to the plant by the AM fungus; however, suppression of premature arbuscule degeneration does not occur in sulphur-deprived mtpt4 plants. The mtpt4 arbuscule phenotype is strongly correlated with shoot N levels. Analyses of an mtpt4-2 sunn-1 double mutant indicates that SUNN, required for N-mediated autoregulation of nodulation, is not involved. Together, the data reveal an unexpected role for N in the regulation of arbuscule lifespan in AM symbiosis.     

丛枝菌根真菌脂类代谢对共生信号调控的响应和反馈

丛枝菌根(AM)真菌是自然生态系统中分布最为广泛的真菌之一,在自然界物质循环和能量流动中发挥着重要作用。经过长期的协同进化,AM真菌和宿主植物之间形成了完美的互惠互利的共生关系,而真菌的脂类代谢可能是揭示共生秘密的关键所在。本文综述了AM真菌脂类代谢在共生关系建立和维持中关键作用的最新研究进展,重点探讨了AM真菌脂类代谢对共生信号调控的响应和反馈机制,主要包括:AM真菌脂类存储和释放对共生和非共生状态的响应,以及脂类代谢产物变化与共生营养传递之间的关系;脂类分解过程在共生建立初期对信号分子调控发生的响应,以及相应的物质转化和能量代谢;菌根共生互惠互利关系维持中,真菌脂类代谢与信号分子交流通道的相互渗透和影响。本文对于理解菌根共生机制,促进菌根在生产中的应用具有促进作用。     

Two Lotus japonicus symbiosis mutants impaired at distinct steps of arbuscule development

Arbuscular mycorrhiza (AM) fungi form nutrient‐acquiring symbioses with the majority of higher plants. Nutrient exchange occurs via arbuscules, highly branched hyphal structures that are formed within root cortical cells. With a view to identifying host genes involved in AM development, we isolated Lotus japonicus AM‐defective mutants via a microscopic screen of an ethyl methanesulfonate‐mutagenized population. A standardized mapping procedure was developed that facilitated positioning of the defective loci on the genetic map of L. japonicus, and, in five cases, allowed identification of mutants of known symbiotic genes. Two additional mutants representing independent loci did not form mature arbuscules during symbiosis with two divergent AM fungal species, but exhibited signs of premature arbuscule arrest or senescence. Marker gene expression patterns indicated that the two mutants are affected in distinct steps of arbuscule development. Both mutants formed wild‐type‐like root nodules upon inoculation with Mesorhizobium loti, indicating that the mutated loci are essential during AM but not during root nodule symbiosis.     

Effect of mutations in the pea genes <Emphasis Type="Italic">Sym33</Emphasis> and <Emphasis Type="Italic">Sym40</Emphasis>

Two symbiotic pea (Pisum sativum L.) mutants SGEFix(-)-1 (sym40) and SGEFix(-)-2 (sym33) with abnormalities in infection thread formation in symbiotic root nodules were characterised with respect to dynamics of arbuscule development at 15 degrees C and 24 degrees C. Mutation of sym33 decreased mycorrhiza colonisation at both temperatures and delayed arbuscule development at 15 degrees C, whereas mutation of sym40 accelerated mycorrhiza colonisation and arbuscule senescence at 24 degrees C. The differences between the mutants and the wild-type were more pronounced at 24 degrees C, a temperature close to the optimum for pea growth. The results demonstrate that both pea genes are important in the control of arbuscular mycorrhiza development and can be considered necessary for the tripartite symbiosis in pea.     

Successful joint ventures of plants: arbuscular mycorrhiza and beyond

Among the oldest symbiotic associations of plants are arbuscular mycorrhiza (AM) with fungi of the phylum Glomeromycota. Although many of the symbiotic signaling components have been identified on the side of the plant, AM fungi have long evaded genetic analysis owing to their strict biotrophy and their exceptional genetics. Recently, the identification of the fungal symbiosis signal (Myc factor) and of a corresponding Myc factor receptor, and new insights into AM fungal genetics, have opened new avenues to address early communication and functional aspects of AM symbiosis. These advances will pave the way for breeding programs towards adapted AM fungi for crop production, and will shed light on the ecology and evolution of this remarkably successful symbiosis.     

Gibberellin regulates infection and colonization of host roots by arbuscular mycorrhizal fungi

Arbuscular mycorrhiza (AM) is established by the entry of AM fungi into the host plant roots and the formation of symbiotic structures called arbuscules. The host plant supplies photosynthetic products to the AM fungi, which in return provide phosphate and other minerals to the host through the arbuscules. Both partners gain great advantages from this symbiotic interaction, and both regulate AM development. Our recent work revealed that gibberellic acids (GAs) are required for AM development in the legume Lotus japonicus. GA signaling interact with symbiosis signaling pathways, directing AM fungal colonization in host roots. Expression analysis showed that genes for GA biosynthesis and metabolism were induced in host roots around AM fungal hyphae, suggesting that the GA signaling changes with both location and time during AM development. The fluctuating GA concentrations sometimes positively and sometimes negatively affect the expression of AM-induced genes that regulate AM fungal infection and colonization.     

Two Medicago truncatula Half-ABC Transporters Are Essential for Arbuscule Development in Arbuscular Mycorrhizal Symbiosis

In the symbiotic association of plants and arbuscular mycorrhizal (AM) fungi, the fungal symbiont resides in the root cortical cells where it delivers mineral nutrients to its plant host through branched hyphae called arbuscules. Here, we report a Medicago truncatula mutant, stunted arbuscule (str), in which arbuscule development is impaired and AM symbiosis fails. In contrast with legume symbiosis mutants reported previously, str shows a wild-type nodulation phenotype. STR was identified by positional cloning and encodes a half-size ATP binding cassette (ABC) transporter of a subfamily (ABCG) whose roles in plants are largely unknown. STR is a representative of a novel clade in the ABCG subfamily, and its orthologs are highly conserved throughout the vascular plants but absent from Arabidopsis thaliana. The STR clade is unusual in that it lacks the taxon-specific diversification that is typical of the ABCG gene family. This distinct phylogenetic profile enabled the identification of a second AM symbiosis-induced half-transporter, STR2. Silencing of STR2 by RNA interference results in a stunted arbuscule phenotype identical to that of str. STR and STR2 are coexpressed constitutively in the vascular tissue, and expression is induced in cortical cells containing arbuscules. STR heterodimerizes with STR2, and the resulting transporter is located in the peri-arbuscular membrane where its activity is required for arbuscule development and consequently a functional AM symbiosis.     

Expression profiling of up-regulated plant and fungal genes in early and late stages of<Emphasis Type="Italic"> Medicago truncatula-Glomus mosseae</Emphasis> interactions

Suppression subtractive hybridization (SSH), expression profiling and EST sequencing identified 12 plant genes and six fungal genes that are expressed in the arbuscular mycorrhizal symbiosis between Medicago truncatula and Glomus mosseae. All the plant genes and three of the fungal genes were up-regulated in symbiotic tissues. Expression of 15 of the genes is described for the first time in mycorrhizal roots and two are novel sequences. Six M. truncatula genes were also activated during appressorium formation at the root surface, suggesting a role in this early stage of mycorrhiza establishment, whilst the other six plant genes were only induced in the late stages of mycorrhization and could be involved in the development or functioning of the symbiosis. Phosphate fertilization had no significant influence on expression of any of the plant genes. Expression profiling of G. mosseae genes indicated that two of them may be associated with appressorium development on roots and one with arbuscule formation or function. The other three fungal genes were expressed throughout the life-cycle of G. mosseae.     

Molecular genetics of the arbuscular mycorrhizal symbiosis

During arbuscular mycorrhiza (AM) development, fungal hyphae grow throughout root epidermal, exodermal and cortical cell layers to reach the inner cortex where the symbiosis' functional units, the arbuscles, develop. Three essential components of a plant signalling network, a receptor-like kinase, a predicted ion-channel and a calmodulin-dependent protein kinase have been identified. A detailed morphological study of symbiotic plant mutants revealed that different subsets of plant genes support the progress of fungal infection in successive root cell layers. Moreover, evidence of a diffusible fungal signalling factor that triggers gene activation in the root has recently been obtained.     

The PAM1 gene of petunia,required for intracellular accommodation and morphogenesis of arbuscular mycorrhizal fungi,encodes a homologue of VAPYRIN

Most terrestrial plants engage into arbuscular mycorrhizal (AM) symbiosis with fungi of the phylum Glomeromycota. The initial recognition of the fungal symbiont results in the activation of a symbiosis signalling pathway that is shared with the root nodule symbiosis (common SYM pathway). The subsequent intracellular accommodation of the fungus, and the elaboration of its characteristic feeding structures, the arbuscules, depends on a genetic programme in the plant that has recently been shown to involve the VAPYRIN gene in Medicaco truncatula. We have previously identified a mutant in Petunia hybrida, penetration and arbuscule morphogenesis 1 (pam1), that is defective in the intracellular stages of AM development. Here, we report on the cloning of PAM1, which encodes a VAPYRIN homologue. PAM1 protein localizes to the cytosol and the nucleus, with a prominent affinity to mobile spherical structures that are associated with the tonoplast, and are therefore referred to as tonospheres. In mycorrhizal roots, tonospheres were observed in the vicinity of intracellular hyphae, where they may play an essential role in the accommodation and morphogenesis of the fungal endosymbiont.     

The Roles of Auxins and Cytokinins in Mycorrhizal Symbioses

Abstract Most land plant species that have been examined exist naturally with a higher fungus living in and around their roots in a symbiotic partnership called a mycorrhiza. Several types of mycorrhizal symbiosis exist, defined by the host/partner combination and the morphology of the symbiotic structures. The arbuscular mycorrhiza (AM) is ancient and may have co-evolved with land plants. Emerging results from gene expression studies have suggested that subsets of AM genes were co-opted during the evolution of other biotrophic symbioses. Here we compare the roles of phytohormones in AM symbiosis and ectomycorrhizas (EC), a more recent symbiosis. To date, there is little evidence of physiologic overlap between the two symbioses with respect to phytohormone involvement. Research on AM has shown that cytokinin (CK) accumulation is specifically enhanced by symbiosis throughout the plant. We propose a pathway of events linking enhanced CK to development of the AM. Additional and proposed involvement of other phytohormones are also described. The role of auxin in EC symbiosis and recent research advances on the topic are reviewed. We have reflected the literature bias in reporting individual growth regulator effects. However, we consider that gradients and ratios of these molecules are more likely to be the causal agents of morphologic changes resulting from fungal associations. We expect that once the individual roles of these compounds are explained, the subtleties of their function will be more clearly addressed.     

NENA, a Lotus japonicus Homolog of Sec13, Is Required for Rhizodermal Infection by Arbuscular Mycorrhiza Fungi and Rhizobia but Dispensable for Cortical Endosymbiotic Development

Legumes form symbioses with arbuscular mycorrhiza (AM) fungi and nitrogen fixing root nodule bacteria. Intracellular root infection by either endosymbiont is controlled by the activation of the calcium and calmodulin-dependent kinase (CCaMK), a central regulatory component of the plant’s common symbiosis signaling network. We performed a microscopy screen for Lotus japonicus mutants defective in AM development and isolated a mutant, nena, that aborted fungal infection in the rhizodermis. NENA encodes a WD40 repeat protein related to the nucleoporins Sec13 and Seh1. Localization of NENA to the nuclear rim and yeast two-hybrid experiments indicated a role for NENA in a conserved subcomplex of the nuclear pore scaffold. Although nena mutants were able to form pink nodules in symbiosis with Mesorhizobium loti, root hair infection was not observed. Moreover, Nod factor induction of the symbiotic genes NIN, SbtM4, and SbtS, as well as perinuclear calcium spiking, were impaired. Detailed phenotypic analyses of nena mutants revealed a rhizobial infection mode that overcame the lack of rhizodermal responsiveness and carried the hallmarks of crack entry, including a requirement for ethylene. CCaMK-dependent processes were only abolished in the rhizodermis but not in the cortex of nena mutants. These data support the concept of tissue-specific components for the activation of CCaMK.     

植物激素调控丛枝菌根发育的作用机制研究进展

丛枝菌根（arbuscular mycorrhiza，AM）是土壤中AM真菌和绝大多数维管植物根系长期进化过程中相互识别、相互作用形成的互利共生体。AM的发育与功能效应依赖AM真菌-寄主植物之间精准的“分子对话”，同时受到环境条件特别是土壤养分水平、干旱和盐渍化的制约。植物激素作为低浓度的小分子有机物，是参与调控AM共生过程的重要信号分子。其中，主要有9种植物激素参与AM发育过程且分工各有不同：独脚金内酯（strigolactones，SLs）参与AM真菌-寄主植物之间最初的共生识别，脱落酸（abscisic acid，ABA）和油菜素内酯（brassinosteroid，BR）促进前期的菌丝入侵，但水杨酸（salicylic acid，SA）和乙烯（ethylene，ET）抑制前期的菌丝入侵，生长素（auxin，Aux）、ABA和BR促进随后的丛枝形成而ET和赤霉素（gibberellin，GA）的作用则相反，茉莉酸（jasmonic acid，JA）对菌丝入侵与丛枝形成均可能存在正调控或负调控作用。目前细胞分裂素（cytokinin，CTK）在AM发育中的作用尚不明确。更为复杂的是，通常植物激素信号之间的交叉互作决定AM的发育进程。本文针对AM发育过程总结了不同植物激素的调控作用特点和不同植物激素信号之间的互作（协同或拮抗），以及胁迫条件下不同植物激素信号的可能调控机制。深入研究和系统阐明植物激素调控AM真菌-寄主植物共生的生理/分子机制，将有助于促进生物共生学理论研究及菌根技术的应用。     

Symbiosis-related pea genes modulate fungal and plant gene expression during the arbuscule stage of mycorrhiza with Glomus intraradices

The arbuscular mycorrhiza association results from a successful interaction between genomes of the plant and fungal symbiotic partners. In this study, we analyzed the effect of inactivation of late-stage symbiosis-related pea genes on symbiosis-associated fungal and plant molecular responses in order to gain insight into their role in the functional mycorrhizal association. The expression of a subset of ten fungal and eight plant genes, previously reported to be activated during mycorrhiza development, was compared in Glomus intraradices-inoculated wild-type and isogenic genotypes of pea mutated for the PsSym36, PsSym33, and PsSym40 genes where arbuscule formation is inhibited or fungal turnover modulated, respectively. Microdissection was used to corroborate arbuscule-related fungal gene expression. Molecular responses varied between pea genotypes and with fungal development. Most of the fungal genes were downregulated when arbuscule formation was defective, and several were upregulated with more rapid fungal development. Some of the plant genes were also affected by inactivation of the PsSym36, PsSym33, and PsSym40 loci, but in a more time-dependent way during root colonization by G. intraradices. Results indicate a role of the late-stage symbiosis-related pea genes not only in mycorrhiza development but also in the symbiotic functioning of arbuscule-containing cells.