Symbiotic root nodules in leguminous plants result from interaction between the plant and nitrogen-fixing rhizobia bacteria. There are two major types of legume nodules, determinate and indeterminate. Determinate nodules do not have a persistent meristem, while indeterminate nodules have a persistent meristem. Auxin is thought to play a role in the development of both these types of nodules. However, inhibition of rootward auxin transport at the site of nodule initiation is crucial for the development of indeterminate nodules but not determinate nodules. Using the synthetic auxin-responsive DR5 promoter in soybean (Glycine max), we show that there is relatively low auxin activity during determinate nodule initiation and that it is restricted to the nodule periphery subsequently during development. To examine if and what role auxin plays in determinate nodule development, we generated soybean composite plants with altered sensitivity to auxin. We overexpressed microRNA393 to silence the auxin receptor gene family, and these roots were hyposensitive to auxin. These roots nodulated normally, suggesting that only minimal/reduced auxin signaling is required for determinate nodule development. We overexpressed microRNA160 to silence a set of repressor auxin response factor transcription factors, and these roots were hypersensitive to auxin. These roots were not impaired in epidermal responses to rhizobia but had significantly reduced nodule primordium formation, suggesting that auxin hypersensitivity inhibits nodule development. These roots were also hyposensitive to cytokinin and had attenuated expression of key nodulation-associated transcription factors known to be regulated by cytokinin. We propose a regulatory feedback loop involving auxin and cytokinin during nodulation.

Ralstonia solanacearum is the causal agent of the devastating bacterial wilt disease, which colonizes susceptible Medicago truncatula via the intact root tip. Infection involves four steps: appearance of root tip symptoms, root tip cortical cell invasion, vessel colonization, and foliar wilting. We examined this pathosystem by in vitro inoculation of intact roots of susceptible or resistant M. truncatula with the pathogenic strain GMI1000. The infection process was type III secretion system dependent and required two type III effectors, Gala7 and AvrA, which were shown to be involved at different stages of infection. Both effectors were involved in development of root tip symptoms, and Gala7 was the main determinant for bacterial invasion of cortical cells. Vessel invasion depended on the host genetic background and was never observed in the resistant line. The invasion of the root tip vasculature in the susceptible line caused foliar wilting. The avrA mutant showed reduced aggressiveness in all steps of the infection process, suggesting a global role in R. solanacearum pathogenicity. The roles of these two effectors in subsequent stages were studied using an assay that bypassed the penetration step; with this assay, the avrA mutant showed no effect compared with the GMI1000 strain, indicating that AvrA is important in early stages of infection. However, later disease symptoms were reduced in the gala7 mutant, indicating a key role in later stages of infection.

BackgroundmicroRNAs (miRNAs) are key regulators of gene expression and play important roles in many aspects of plant biology. The role(s) of miRNAs in nitrogen-fixing root nodules of leguminous plants such as soybean is not well understood. We examined a library of small RNAs from Bradyrhizobium japonicum-inoculated soybean roots and identified novel miRNAs. In order to enhance our understanding of miRNA evolution, diversification and function, we classified all known soybean miRNAs based on their phylogenetic conservation (conserved, legume- and soybean-specific miRNAs) and examined their genome organization, family characteristics and target diversity. We predicted targets of these miRNAs and experimentally validated several of them. We also examined organ-specific expression of selected miRNAs and their targets.ResultsWe identified 120 previously unknown miRNA genes from soybean including 5 novel miRNA families. In the soybean genome, genes encoding miRNAs are primarily intergenic and a small percentage were intragenic or less than 1000 bp from a protein-coding gene, suggesting potential co-regulation between the miRNA and its parent gene. Difference in number and orientation of tandemly duplicated miRNA genes between orthologous genomic loci indicated continuous evolution and diversification. Conserved miRNA families are often larger in size and produce less diverse mature miRNAs than legume- and soybean-specific families. In addition, the majority of conserved and legume-specific miRNA families produce 21 nt long mature miRNAs with distinct nucleotide distribution and regulate a more conserved set of target mRNAs compared to soybean-specific families. A set of nodule-specific target mRNAs and their cognate regulatory miRNAs had inverse expression between root and nodule tissues suggesting that spatial restriction of target gene transcripts by miRNAs might govern nodule-specific gene expression in soybean.ConclusionsGenome organization of soybean miRNAs suggests that they are actively evolving. Distinct family characteristics of soybean miRNAs suggest continuous diversification of function. Inverse organ-specific expression between selected miRNAs and their targets in the roots and nodules, suggested a potential role for these miRNAs in regulating nodule development.

Auxin action is mediated by a complex signalling pathway involving transcription factors of the auxin response factor (ARF) family. In Arabidopsis, microRNA160 (miR160) negatively regulates three ARF genes (ARF10/ARF16/ARF17) and therefore controls several developmental processes, including primary and lateral root growth. Here, we analysed the role of miR160 in root development and nodulation in Medicago truncatula Gaertn. Bioinformatic analyses identified two main mtr-miR160 variants (mtr-miR160abde and mtr-miR160c) and 17 predicted ARF targets. The miR160-dependent cleavage of four predicted targets in roots was confirmed by analysis of parallel analysis of RNA ends (PARE) data and RACE-PCR experiments. Promoter-GUS analyses for mtr-miR160d and mtr-miR160c genes revealed overlapping but distinct expression profiles during root and nodule development. In addition, the early miR160 activation in roots during symbiotic interaction was not observed in mutants of the nodulation signalling or autoregulation pathways. Composite plants that overexpressed mtr-miR160a under two different promoters exhibited distinct defects in root growth and nodulation: the p35S:miR160a construct led to reduced root length associated to a severe disorganisation of the RAM, whereas pCsVMV:miR160a roots showed gravitropism defects and lower nodule numbers. Our results suggest that a regulatory loop involving miR160/ARFs governs root and nodule organogenesis in M. truncatula.

Ralstonia solanacearum, the causal agent of bacterial wilt, exerts its pathogenicity through more than a hundred secreted proteins, many of them depending directly on the functionality of a type 3 secretion system. To date, only few type 3 effectors have been identified as required for bacterial pathogenicity, notably because of redundancy among the large R. solanacearum effector repertoire. In order to identify groups of effectors collectively promoting disease on susceptible hosts, we investigated the role of putative post-translational regulators in the control of type 3 secretion. A shotgun secretome analysis with label-free quantification using tandem mass spectrometry was performed on the R. solanacearum GMI1000 strain. There were 228 proteins identified, among which a large proportion of type 3 effectors, called Rip (Ralstonia injected proteins). Thanks to this proteomic approach, RipBJ was identified as a new effector specifically secreted through type 3 secretion system and translocated into plant cells. A focused Rip secretome analysis using hpa (hypersensitive response and pathogenicity associated) mutants revealed a fine secretion regulation and specific subsets of Rips with different secretion patterns. We showed that a set of Rips (RipF1, RipW, RipX, RipAB, and RipAM) are secreted in an Hpa-independent manner. We hypothesize that these Rips could be preferentially involved in the first stages of type 3 secretion. In addition, the secretion of about thirty other Rips is controlled by HpaB and HpaG. HpaB, a candidate chaperone was shown to positively control secretion of numerous Rips, whereas HpaG was shown to act as a negative regulator of secretion. To evaluate the impact of altered type 3 effectors secretion on plant pathogenesis, the hpa mutants were assayed on several host plants. HpaB was required for bacterial pathogenicity on multiple hosts whereas HpaG was found to be specifically required for full R. solanacearum pathogenicity on the legume plant Medicago truncatula. Molecular & Cellular Proteomics 15: 10.1074/ mcp.M115.051078, 598-613, 2016.The soil-borne vascular bacterium R. solanacearum is described as one of the most destructive plant pathogenic bacterium worldwide (1), mainly because of its broad host range and wide geographic distribution. Indeed, R. solanacearum attacks more than 250 plant species, distributed in more than 50 botanical families (2). R. solanacearum causes dramatic crop losses, more specifically in the tropics, notably affecting emerging countries. Durable protection strategies against this bacterium are lacking. R. solanacearum penetrates into the plant via the roots, and then colonizes the xylem vessels. The bacterium reaches the aerial parts of the plant, causes wilting symptoms leading to the death of the plant, and will eventually return to the soil, completing the cycle (3). To achieve these first colonization steps, the bacterium uses a molecular syringe called the type 3 secretion system (T3SS), 1 which deFrom the ‡INRA, Laboratoire des Interactions Plantes-...

The roles of auxin in the regulation of symbiotic legume nodule formation are unclear. We recently showed that enhanced sensitivity to auxin resulting from overexpression of miR160 inhibits determinate nodule formation in soybean. We examined the roles of miR393 and miR164 in soybean (that forms determinate nodules) and Medicago truncatula (that forms indeterminate nodules). Our results together with previous studies suggest that indeterminate nodule formation requires a higher, but narrow window of auxin sensitivity and that miR164 regulation is not crucial for determinate nodule formation.

We recently reported that hairpin (or stem-loop) priming is better-suited than polyA tailing to generate cDNA for plant microRNA qPCR. One major limitation of this method is the need to perform individual cDNA synthesis reactions for the reference gene and test miRNAs. Here, we report a novel fusion primer that allows multiplexed hairpin cDNA synthesis of the most-commonly used reference gene, nucleolar small RNA U6, together with test miRNAs. We also propose the use of miR1515 as a house keeping control for tropical legumes. We show that multiplexed cDNA synthesis does not result in loss of sensitivity and reduces the amount of RNA required for miRNA gene expression assays.

Many pathogenic bacteria have evolved a type III secretion system (T3SS) to successfully invade their host. This extracellular apparatus allows the translocation of proteins, called type III effectors (T3Es), directly into the host cells. T3Es are virulence factors that have been shown to interfere with the host's immunity or to provide nutrients from the host to the bacteria. The Gram-negative bacterium Ralstonia solanacearum is a worldwide major crop pest whose virulence strongly relies on the T3SS. In R. solanacearum, transcriptional regulation has been extensively studied. However, very few data are available concerning the role played by type III-associated regulators, such as type III chaperones and T3SS control proteins. Here, we characterized HpaP, a putative type III secretion substrate specificity switch (T3S4) protein of R. solanacearum which is not secreted by the bacterium or translocated in the plant cells. HpaP self-interacts and interacts with the PopP1 T3E. HpaP modulates the secretion of early (HrpY pilin) and late (AvrA and PopP1 T3Es) type III substrates. HpaP is dispensable for the translocation of T3Es into the host cells. Finally, we identified two regions of five amino acids in the T3S4 domain that are essential for efficient PopP1 secretion and for HpaP's role in virulence on tomato and Arabidopsis thaliana, but not required for HpaP-HpaP and HpaP-PopP1 interactions. Taken together, our results indicate that HpaP is a putative R. solanacearum T3S4 protein important for full pathogenicity on several hosts, acting as a helper for PopP1 secretion, and repressing AvrA and HrpY secretion.