Assoc.Prof. Panlada Tittabutr

Year	Title	Journal
2018	Regulation of nitrogen fixation in Bradyrhizobium sp. Strain DOA9 involves two distinct NifA regulatory proteins that are functionally redundant during symbiosis but not during free-living growth Wongdee J., Boonkerd N., Teaumroong N., Tittabutr P., Giraud E.	Frontiers in Microbiology
Abstract: The Bradyrhizobium sp. DOA9 strain displays the unusual properties to have a symbiotic plasmid and to fix nitrogen during both free-living and symbiotic growth. Sequence genome analysis shows that this strain contains the structural genes of dinitrogenase (nifDK) and the nifA regulatory gene on both the plasmid and chromosome. It was previously shown that both nifDK clusters are differentially expressed depending on growth conditions, suggesting different mechanisms of regulation. In this study, we examined the functional regulatory role of the two nifA genes found on the plasmid (nifAp) and chromosome (nifAc) that encode proteins with a moderate level of identity (55%) and different structural architectures. Using gusA (β-glucuronidase) reporter strains, we showed that both nifA genes were expressed during both the free-living and symbiotic growth stages. During symbiosis with Aeschynomene americana, mutants in only one nifA gene were not altered in their symbiotic properties, while a double nifA mutant was drastically impaired in nitrogen fixation, indicating that the two NifA proteins are functionally redundant during this culture condition. In contrast, under in vitro conditions, the nifAc mutant was unable to fix nitrogen, and no effect of the nifAp mutation was detected, indicating that NifAc is essential to activate nif genes during free-living growth. In accordance, the nitrogenase fixation deficiency of this mutant could be restored by the introduction of nifAc but not by nifAp or by two chimeric nifA genes encoding hybrid proteins with the N-terminus part of NifAc and the C-terminus of NifAp. Furthermore, transcriptional analysis by RT-qPCR of the WT and two nifA mutant backgrounds showed that NifAc and NifAp activated the expression of both chromosome and plasmid structural nifDK genes during symbiosis, while only NifAc activated the expression of nifDKc during free-living conditions. In summary, this study provides a better overview of the complex mechanisms of regulation of the nitrogenase genes in the DOA9 strain that involve two distinct NifA proteins, which are exchangeable during symbiosis for the activation of nif genes but not during free-living growth where NifAc is essential for the activation of nifDKc. © 2018 Wongdee, Boonkerd, Teaumroong, Tittabutr and Giraud. Keyword: Bradyrhizobium; Legume; NifA; Nitrogen; Nitrogenase; Rhizobium; Symbiosis Scopus Link: https://www.scopus.com/inward/record.uri?eid=2-s2.0-85050494484&doi=10.3389%2ffmicb.2018.01644&partnerID=40&md5=64a3e1a5e06dc537c4f1419153bcd8a8 DOI: 10.3389/fmicb.2018.01644
2018	Brevibacillus sp. Promotes maize root colonization by acaulospora tuberculata and the alteration of associated plant protein responses Yuttavanichakul W., Teamtisong K., Teaumroong N., Boonkerd N., Tittabutr P.	Journal of Plant Interactions
Abstract: Abilities of rhizobacteria to promote mycorrhization of arbuscular mycorrhiza (AM) on maize roots have been documented. In this study, the rhizospheric bacterium, Brevibacillus sp. SUT47 was found to significantly promote spore number and root colonization of Acaulospora tuberculata. To understand how maize roots respond to SUT47, a comparative proteomics analysis was performed. We found that at 30 days after inoculation (dai), the proteins involved in plant defense mechanism and Reactive Oxygen Species (ROS)-scavenging enzymes were the main proteins altered in tested maize roots. Levels of salicylic acid, hydrogen peroxide, and the activity of superoxide dismutase were significantly decreased in AM+SUT47 roots at 7 dai, while the activities of peroxidase and ascorbate peroxidase increased especially in AM+SUT47 roots at 30 dai. Thus, this work showed the alteration of some plant defense-related compounds and antioxidative enzyme activities that are associated with an enhancing maize root colonization by AM when co-inoculated with SUT47. © 2018 The Author(s). Keyword: Antioxidative enzymes; Arbuscular mycorrhizal fungi; Maize root colonization; PGPR; Plant defense-related compounds; Protein changes Scopus Link: https://www.scopus.com/inward/record.uri?eid=2-s2.0-85064120669&doi=10.1080%2f17429145.2018.1547844&partnerID=40&md5=21fe488b7c90345fd99a2ce1413f8122 DOI: 10.1080/17429145.2018.1547844
2017	Genome sequence of Bacillus velezensis S141, a new strain of plant growthpromoting rhizobacterium isolated from soybean rhizosphere Sibponkrung S., Kondo T., Tanaka K., Tittabutr P., Boonkerd N., Teaumroong N., Yoshida K.-I.	Genome Announcements
Abstract: Bacillus velezensis strain S141 is a plant growth-promoting rhizobacterium isolated from soybean (Glycine max) rhizosphere that enhances soybean growth, nodulation, and N2 fixation efficiency by coinoculation with Bradyrhizobium diazoefficiens USDA110. The S141 genome was identified to comprise a 3,974,582-bp-long circular DNA sequence encoding at least 3,817 proteins. © 2017 Sibponkrung et al. Keyword: Scopus Link: https://www.scopus.com/inward/record.uri?eid=2-s2.0-85035784492&doi=10.1128%2fgenomeA.01312-17&partnerID=40&md5=bba54824eb41e6d37b79a97d60ac0909 DOI: 10.1128/genomeA.01312-17
2017	Potential of rice stubble as a reservoir of bradyrhizobial inoculum in rice-legume crop rotation Piromyou P., Greetatorn T., Teamtisong K., Tittabutr P., Boonkerd N., Teaumroong N.	Applied and Environmental Microbiology
Abstract: Bradyrhizobium encompasses a variety of bacteria that can live in symbiotic and endophytic associations with leguminous and nonleguminous plants, such as rice. Therefore, it can be expected that rice endophytic bradyrhizobia can be applied in the rice-legume crop rotation system. Some endophytic bradyrhizobial strains were isolated from rice (Oryza sativa L.) tissues. The rice biomass could be enhanced when supplementing bradyrhizobial strain inoculation with KNO3, NH4NO3, or urea, especially in Bradyrhizobium sp. strain SUTN9-2. In contrast, the strains which suppressed rice growth were photosynthetic bradyrhizobia and were found to produce nitric oxide (NO) in the rice root. The expression of genes involved in NO production was conducted using a quantitative reverse transcription-PCR (qRT-PCR) technique. The nirK gene expression level in Bradyrhizobium sp. strain SUT-PR48 with nitrate was higher than that of the norB gene. In contrast, the inoculation of SUTN9-2 resulted in a lower expression of the nirK gene than that of the norB gene. These results suggest that SUT-PR48 may accumulate NO more than SUTN9-2 does. Furthermore, the nifH expression of SUTN9-2 was induced in treatment without nitrogen supplementation in an endophytic association with rice. The indole-3-acetic acid (IAA) and 1-amino-cyclopropane-1-carboxylic acid (ACC) deaminase produced in planta by SUTN9-2 were also detected. Enumeration of rice endophytic bradyrhizobia from rice tissues revealed that SUTN9-2 persisted in rice tissues until rice-harvesting season. The mung bean (Vigna radiata) can be nodulated after rice stubbles were decomposed. Therefore, it is possible that rice stubbles can be used as an inoculum in the rice-legume crop rotation system under both low- and high-organic-matter soil conditions. © 2017 American Society for Microbiology. Keyword: Mung bean; Rice endophytic bradyrhizobia; Rice-legume crop rotation Scopus Link: https://www.scopus.com/inward/record.uri?eid=2-s2.0-85032679872&doi=10.1128%2fAEM.01488-17&partnerID=40&md5=90eb6055340309ae688dac689810c12d DOI: 10.1128/AEM.01488-17
2017	Type 3 secretion system (T3SS) of Bradyrhizobium sp. DOA9 and its roles in legume symbiosis and rice endophytic association Songwattana P., Noisangiam R., Teamtisong K., Prakamhang J., Teulet A., Tittabutr P., Piromyou P., Boonkerd N., Giraud E., Teaumroong N.	Frontiers in Microbiology
Abstract: The Bradyrhizobium sp. DOA9 strain isolated from a paddy field has the ability to nodulate a wide spectrum of legumes. Unlike other bradyrhizobia, this strain has a symbiotic plasmid harboring nod, nif, and type 3 secretion system (T3SS) genes. This T3SS cluster contains all the genes necessary for the formation of the secretory apparatus and the transcriptional activator (TtsI), which is preceded by a nod-box motif. An in silico search predicted 14 effectors putatively translocated by this T3SS machinery. In this study, we explored the role of the T3SS in the symbiotic performance of DOA9 by evaluating the ability of a T3SS mutant (ΩrhcN) to nodulate legumes belonging to Dalbergioid, Millettioid, and Genistoid tribes. Among the nine species tested, four (Arachis hypogea, Vigna radiata, Crotalaria juncea, and Macroptilium atropurpureum) responded positively to the rhcN mutation (ranging from suppression of plant defense reactions, an increase in the number of nodules and a dramatic improvement in nodule development and infection), one (Stylosanthes hamata) responded negatively (fewer nodules and less nitrogen fixation) and four species (Aeschynomene americana, Aeschynomene afraspera, Indigofera tinctoria, and Desmodium tortuosum) displayed no phenotype. We also tested the role of the T3SS in the ability of the DOA9 strain to endophytically colonize rice roots, but detected no effect of the T3SS mutation, in contrast to what was previously reported in the Bradyrhizobium SUTN9-2 strain. Taken together, these data indicate that DOA9 contains a functional T3SS that interferes with the ability of the strain to interact symbiotically with legumes but not with rice. © 2017 Songwattana, Noisangiam, Teamtisong, Prakamhang, Teulet, Tittabutr, Piromyou, Boonkerd, Giraud and Teaumroong. Keyword: Bradyrhizobium; Effector proteins; Legume; Symbiosis; Type 3 secretion system Scopus Link: https://www.scopus.com/inward/record.uri?eid=2-s2.0-85029772063&doi=10.3389%2ffmicb.2017.01810&partnerID=40&md5=f9c8fca735194dbb693f071b8b84f026 DOI: 10.3389/fmicb.2017.01810
2017	Generation of a rabbit single-chain fragment variable (scFv) antibody for specific detection of Bradyrhizobium sp. DOA9 in both free-living and bacteroid forms Vu N.X., Pruksametanan N., Srila W., Yuttavanichakul W., Teamtisong K., Teaumroong N., Boonkerd N., Tittabutr P., Yamabhai M.	PLoS ONE
Abstract: A simple and reliable method for the detection of specific nitrogen-fixing bacteria in both free-living and bacteroid forms is essential for the development and application of biofertilizer. Traditionally, a polyclonal antibody generated from an immunized rabbit was used for detection. However, the disadvantages of using a polyclonal antibody include limited supply and cross-reactivity to related bacterial strains. This is the first report on the application of phage display technology for the generation of a rabbit recombinant monoclonal antibody for specific detection and monitoring of nitrogen-fixing bacteria in both free-living form and in plant nodules. Bradyrhizobium sp. DOA9, a broad host range soil bacteria, originally isolated from the root nodules of Aeschynomene americana in Thailand was used as a model in this study. A recombinant single-chain fragment variable (scFv) antibody library was constructed from the spleen of a rabbit immunized with DOA9. After three rounds of biopanning, one specific phage-displayed scFv antibody, designated bDOA9rb8, was identified. Specific binding of this antibody was confirmed by phage enzyme-linked immunosorbent assay (phage ELISA). The phage antibody could bind specifically to DOA9 in both free-living cells (pure culture) and bacteroids inside plant nodules. In addition to phage ELISA, specific and robust immunofluorescence staining of both free-living and bacteroid forms could also be observed by confocal-immunofluorescence imaging, without cross-reactivity with other tested bradyrhizobial strains. Moreover, specific binding of free scFv to DOA9 was also demonstrated by ELISA. This recombinant antibody can also be used for the study of the molecular mechanism of plant-microbe interactions in the future. © 2017 Vu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Keyword: Scopus Link: https://www.scopus.com/inward/record.uri?eid=2-s2.0-85021681241&doi=10.1371%2fjournal.pone.0179983&partnerID=40&md5=be068b16868a1805f50a5ad0a68f8aaa DOI: 10.1371/journal.pone.0179983
2016	NifDK clusters located on the chromosome and megaplasmid of bradyrhizobium sp. Strain DOA9 contribute differently to nitrogenase activity during symbiosis and free-living growth Wongdee J., Songwattana P., Nouwen N., Noisangiam R., Fardoux J., Chaintreuil C., Teaumroong N., Tittabutr P., Giraud E.	Molecular Plant-Microbe Interactions
Abstract: Bradyrhizobium sp. strain DOA9 contains two copies of the nifDK genes, nifDKc, located on the chromosome, and nifDKp, located on a symbiotic megaplasmid. Unlike most rhizobia, this bacterium displays nitrogenase activity under both free-living and symbiotic conditions. Transcriptional analysis using gusA reporter strains showed that both nifDK operons were highly expressed under symbiosis, whereas nifDKc was the most abundantly expressed under free-living conditions. During free-living growth, the nifDKp mutation did not affect nitrogenase activity, whereas nitrogenase activity was drastically reduced with the nifDKc mutant. This led us to suppose that nifDKc is the main contributor of nitrogenase activity in the free-living state. In contrast, during symbiosis, no effect of the nifDKc mutation was observed and the nitrogen-fixation efficiency of plants inoculated with the nifDKp mutant was reduced. This suggests that nifDKp plays the main role in nitrogenase enzyme activity during symbiosis. Together, these data suggest that Bradyrhizobiumsp. strain DOA9 contains two functional copies of nifDK genes that are regulated differently and that, depending on their lifestyle, contribute differently to nitrogenase activity. © 2016 The American Phytopathological Society. Keyword: Scopus Link: https://www.scopus.com/inward/record.uri?eid=2-s2.0-84994666900&doi=10.1094%2fMPMI-07-16-0140-R&partnerID=40&md5=58e5ad3c0c6e8150b7f6de9fe316d290 DOI: 10.1094/MPMI-07-16-0140-R
2016	The evolutionary dynamics of ancient and recent polyploidy in the African semiaquatic species of the legume genus Aeschynomene Chaintreuil C., Gully D., Hervouet C., Tittabutr P., Randriambanona H., Brown S.C., Lewis G.P., Bourge M., Cartieaux F., Boursot M., Ramanankierana H., D'Hont A., Teaumroong N., Giraud E., Arrighi J.-F.	New Phytologist
Abstract: The legume genus Aeschynomene is notable in the ability of certain semiaquatic species to develop nitrogen-fixing stem nodules. These species are distributed in two clades. In the first clade, all the species are characterized by the use of a unique Nod-independent symbiotic process. In the second clade, the species use a Nod-dependent symbiotic process and some of them display a profuse stem nodulation as exemplified in the African Aeschynomene afraspera. To facilitate the molecular analysis of the symbiotic characteristics of such legumes, we took an integrated molecular and cytogenetic approach to track occurrences of polyploidy events and to analyze their impact on the evolution of the African species of Aeschynomene. Our results revealed two rounds of polyploidy: a paleopolyploid event predating the African group and two neopolyploid speciations, along with significant chromosomal variations. Hence, we found that A. afraspera (8x) has inherited the contrasted genomic properties and the stem-nodulation habit of its parental lineages (4x). This study reveals a comprehensive picture of African Aeschynomene diversification. It notably evidences a history that is distinct from the diploid Nod-independent clade, providing clues for the identification of the specific determinants of the Nod-dependent and Nod-independent symbiotic processes, and for comparative analysis of stem nodulation. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust. Keyword: Aeschynomene; dysploidy; genome downsizing; polyploidy; stem nodulation; symbiosis Scopus Link: https://www.scopus.com/inward/record.uri?eid=2-s2.0-84963853012&doi=10.1111%2fnph.13956&partnerID=40&md5=15c7a09cac3de52f99891a4500ec6003 DOI: 10.1111/nph.13956
2016	Origin and evolution of nitrogen fixation genes on symbiosis Islands and plasmid in Bradyrhizobium Okubo T., Piromyou P., Tittabutr P., Teaumroong N., Minamisawa K.	Microbes and Environments
Abstract: The nitrogen fixation (nif) genes of nodule-forming Bradyrhizobium strains are generally located on symbiosis islands or symbiosis plasmids, suggesting that these genes have been transferred laterally. The nif genes of rhizobial and non-rhizobial Bradyrhizobium strains were compared in order to infer the evolutionary histories of nif genes. Based on all codon positions, the phylogenetic tree of concatenated nifD and nifK sequences showed that nifDK on symbiosis islands formed a different clade from nifDK on non-symbiotic loci (located outside of symbiosis islands and plasmids) with elongated branches; however, these genes were located in close proximity, when only the 1st and 2nd codon positions were analyzed. The guanine (G) and cytosine (C) content of the 3rd codon position of nifDK on symbiosis islands was lower than that on non-symbiotic loci. These results suggest that nif genes on symbiosis islands were derived from the non-symbiotic loci of Bradyrhizobium or closely related strains and have evolved toward a lower GC content with a higher substitution rate than the ancestral state. Meanwhile, nifDK on symbiosis plasmids clustered with nifDK on non-symbiotic loci in the tree representing all codon positions, and the GC content of symbiotic and non-symbiotic loci were similar. These results suggest that nif genes on symbiosis plasmids were derived from the non-symbiotic loci of Bradyrhizobium and have evolved with a similar evolutionary pattern and rate as the ancestral state. © 2016, Microbes and Environments. All Rights Reserved. Keyword: Bradyrhizobium; Evolution; Guanine and cytosine content; Nitrogen fixation; Nodule symbiosis Scopus Link: https://www.scopus.com/inward/record.uri?eid=2-s2.0-84989211024&doi=10.1264%2fjsme2.ME15159&partnerID=40&md5=74b02d5d41f7046501522ec787f73b83 DOI: 10.1264/jsme2.ME15159
2016	Rhizobium-legume symbiosis in the absence of Nod factors: Two possible scenarios with or without the T3SS Okazaki S., Tittabutr P., Teulet A., Thouin J., Fardoux J., Chaintreuil C., Gully D., Arrighi J.-F., Furuta N., Miwa H., Yasuda M., Nouwen N., Teaumroong N., Giraud E.	ISME Journal
Abstract: The occurrence of alternative Nod factor (NF)-independent symbiosis between legumes and rhizobia was first demonstrated in some Aeschynomene species that are nodulated by photosynthetic bradyrhizobia lacking the canonical nodABC genes. In this study, we revealed that a large diversity of non-photosynthetic bradyrhizobia, including B. elkanii, was also able to induce nodules on the NF-independent Aeschynomene species, A. indica. Using cytological analysis of the nodules and the nitrogenase enzyme activity as markers, a gradient in the symbiotic interaction between bradyrhizobial strains and A. indica could be distinguished. This ranged from strains that induced nodules that were only infected intercellularly to rhizobial strains that formed nodules in which the host cells were invaded intracellularly and that displayed a weak nitrogenase activity. In all non-photosynthetic bradyrhizobia, the type III secretion system (T3SS) appears required to trigger nodule organogenesis. In contrast, genome sequence analysis revealed that apart from a few exceptions, like the Bradyrhizobium ORS285 strain, photosynthetic bradyrhizobia strains lack a T3SS. Furthermore, analysis of the symbiotic properties of an ORS285 T3SS mutant revealed that the T3SS could have a positive or negative role for the interaction with NF-dependent Aeschynomene species, but that it is dispensable for the interaction with all NF-independent Aeschynomene species tested. Taken together, these data indicate that two NF-independent symbiotic processes are possible between legumes and rhizobia: one dependent on a T3SS and one using a so far unknown mechanism. © 2016 International Society for Microbial Ecology. Keyword: Scopus Link: https://www.scopus.com/inward/record.uri?eid=2-s2.0-84936972674&doi=10.1038%2fismej.2015.103&partnerID=40&md5=4a7f34619d2ceda431b82561fd65f140 DOI: 10.1038/ismej.2015.103