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Abstract Background Plant microbiome confers versatile functional roles to enhance survival fitness as well as productivity. In the present study two pearl millet panicle microbiome member species Bacillus subtilis PBs 12 and Bacillus paralicheniformis PBl 36 found to have beneficial traits including plant growth promotion and broad-spectrum antifungal activity towards taxonomically diverse plant pathogens. Understanding the genomes will assist in devising a bioformulation for crop protection while exploiting their beneficial functional roles. Results Two potential firmicute species were isolated from pearl millet panicles. Morphological, biochemical, and molecular characterization revealed their identities as Bacillus subtilis PBs 12 and Bacillus paralicheniformis PBl 36. The seed priming assays revealed the ability of both species to enhance plant growth promotion and seedling vigour index. Invitro assays with PBs 12 and PBl 36 showed the antibiosis effect against taxonomically diverse plant pathogens (Magnaporthe grisea; Sclerotium rolfsii; Fusarium solani; Alternaria alternata; Ganoderma sp.) of crops and multipurpose tree species. The whole genome sequence analysis was performed to unveil the genetic potential of these bacteria for plant protection. The complete genomes of PBs 12 and PBl 36 consist of a single circular chromosome with a size of 4.02 and 4.33 Mb and 4,171 and 4,606 genes, with a G + C content of 43.68 and 45.83%, respectively. Comparative Average Nucleotide Identity (ANI) analysis revealed a close similarity of PBs 12 and PBl 36 with other beneficial strains of B. subtilis and B. paralicheniformis and found distant from B. altitudinis, B. amyloliquefaciens, and B. thuringiensis. Functional annotation revealed a majority of pathway classes of PBs 12 (30) and PBl 36 (29) involved in the biosynthesis of secondary metabolites, polyketides, and non-ribosomal peptides, followed by xenobiotic biodegradation and metabolism (21). Furthermore, 14 genomic regions of PBs 12 and 15 of PBl 36 associated with the synthesis of RiPP (Ribosomally synthesized and post-translationally modified peptides), terpenes, cyclic dipeptides (CDPs), type III polyketide synthases (T3PKSs), sactipeptides, lanthipeptides, siderophores, NRPS (Non-Ribosomal Peptide Synthetase), NRP-metallophone, etc. It was discovered that these areas contain between 25,458 and 33,000 secondary metabolite-coding MiBiG clusters which code for a wide range of products, such as antibiotics. The PCR-based screening for the presence of antimicrobial peptide (cyclic lipopeptide) genes in PBs 12 and 36 confirmed their broad-spectrum antifungal potential with the presence of spoVG, bacA, and srfAA AMP genes, which encode antimicrobial compounds such as subtilin, bacylisin, and surfactin. Conclusion The combined in vitro studies and genome analysis highlighted the antifungal potential of pearl millet panicle-associated Bacillus subtilis PBs12 and Bacillus paralicheniformis PBl36. The genetic ability to synthesize several antimicrobial compounds indicated the industrial value of PBs 12 and PBl 36, which shed light on further studies to establish their action as a biostimulant for crop protection.

Abstract Long non-coding RNAs (lncRNAs) are abundant in plants, however, their regulatory roles remain unclear in most biological processes, such as response in salinity stress which is harm to plant production. Here we show a lncRNA in Medicago truncatula identified from salt-treated Medicago truncatula is important for salinity tolerance. We name the lncRNA LAL, L ncRNA A NTISENSE to M. truncatula L IGHT-HARVESTING CHLOROPHYLL A/B BINDING (MtLHCB) genes. LAL is an antisense to four consecutive MtLHCB genes on chromosome 6. In salt-treated M. truncatula, LAL is suppressed in an early stage but induced later; this pattern is opposite to that of the four MtLHCBs. The lal mutants show enhanced salinity tolerance, while overexpressing LAL disrupts this superior tolerance in the lal background, which indicates its regulatory role in salinity response. The regulatory role of LAL on MtLHCB1.4 is further verified by transient co-expression of LAL and MtLHCB1.4-GFP in tobacco leaves, in which the cleavage of MtLHCB1.4 and production of secondary interfering RNA is identified. This work demonstrates a lncRNA, LAL, functioning as a regulator that fine-tunes salinity tolerance via regulating MtLHCB1s’ expression in M. truncatula.

Abstract Vanadium (V) pollution potentially threatens human health. Here, it is found that nsp1 and nsp2, Rhizobium symbiosis defective mutants of Medicago truncatula, are sensitive to V. Concentrations of phosphorus (P), iron (Fe), and sulfur (S) with V are negatively correlated in the shoots of wild‐type R108, but not in mutant nsp1 and nsp2 shoots. Mutations in the P transporter PHT1, PHO1, and VPT families, Fe transporter IRT1, and S transporter SULTR1/3/4 family confer varying degrees of V tolerance on plants. Among these gene families, MtPT1, MtZIP6, MtZIP9, and MtSULTR1; 1 in R108 roots are significantly inhibited by V stress, while MtPHO1; 2, MtVPT2, and MtVPT3 are significantly induced. Overexpression of Arabidopsis thaliana VPT1 or M. truncatula MtVPT3 increases plant V tolerance. However, the response of these genes to V is weakened in nsp1 or nsp2 and influenced by soil microorganisms. Mutations in NSPs reduce rhizobacterial diversity under V stress and simplify the V‐responsive operational taxonomic unit modules in co‐occurrence networks. Furthermore, R108 recruits more beneficial rhizobacteria related to V, P, Fe, and S than does nsp1 or nsp2. Thus, NSPs can modulate the accumulation and tolerance of legumes to V through P, Fe, and S transporters, ion homeostasis, and rhizobacterial community responses.

Medicago truncatula is a chosen model for legumes towards deciphering fundamental legume biology, especially symbiotic nitrogen fixation. Current genomic resources for M. truncatula include a completed whole genome sequence information for R108 and Jemalong A17 accessions along with the sparse draft genome sequences for other 226 M. truncatula accessions. These genomic resources are complemented by the availability of mutant resources such as retrotransposon (Tnt1) insertion mutants in R108 and fast neutron bombardment (FNB) mutants in A17. In addition, several M. truncatula databases such as small secreted peptides (SSPs) database, transporter protein database, gene expression atlas, proteomic atlas, and metabolite atlas are available to the research community. This review describes these resources and provide information regarding how to access these resources.

Sheath blight is a serious rice disease worldwide, and genes involved in resistance remain unclear. In the present study, a virulent field isolate of Rhizoctonia solani was used to inoculate detached leaves of a sheath blight-resistant rice cultivar, Jasmine 85; a suppression subtractive cDNA library was constructed using RNA isolated 16 h postinoculation (hpi); and differentially expressed genes were identified from the cDNA library. A total of 159 uniquely expressed sequence tags were identified, including 105 from rice with enrichment in categories related to cellular response, molecular signaling, and host defense. Coupled with gene expression studies by DNA microarray, 27 highly induced genes involved in signal transduction and defense responses were identified within 16 hpi. Three members of the ATP-binding cassette (ABC) transporter gene family (OsABC1, OsABC9, and OsABC12) encoding pleiotropic drug resistance-like ABC transporters were mapped to different sheath blight resistance quantitative trait loci (QTLs), and their differential expressions were validated. Three high-resolution melting (HRM) markers were developed from these ABC gene family members to distinguish alleles between sheath blight-susceptible cultivar Lemont and resistant cultivar Jasmine 85. Association of sheath blight resistance to these HRM markers was examined in 77 recombinant inbred lines derived from the cross between Jasmine 85 and Lemont. The OsABC9 gene located in a major sheath blight resistance QTL qShB9-2 showed a major contribution to sheath blight resistance. These results are useful for marker-assisted selection and functional validation of the ABC genes in sheath blight disease resistance. [Figure: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 “No Rights Reserved” license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law, 2022.

Agrobacterium infection can cause defense responses in many plants, which leads to transformation recalcitrance. Here, the authors express type III secretion system in Agrobacterium to deliver effector proteins into plant cells to suppress host defense responses and thus enhance transformation in some plant species.

E3 ubiquitin ligase salt- and drought-induced ring finger 1 (SDIR1) plays a novel role in modulating plant immunity against pathogens. The molecular interactors of SDIR1 during pathogen infection are not known. SDIR1-interacting jasmonate zinc-finger inflorescence meristem domain (JAZ) proteins were identified through a yeast two-hybrid (Y2H) screen. Full-length JAZ9 interacts with SDIR1 only in the presence of coronatine (a bacteria-secreted toxin) or jasmonic acid (JA) in a Y2H assay. The bimolecular fluorescence complementation and pull-down assays confirm the in planta interaction of these proteins. JAZ9 proteins, negative regulators of JA-mediated plant defense, were degraded during the pathogen infection by SDIR1 through a proteasomal pathway causing disease susceptibility against hemibiotrophic pathogens.[Graphic: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 “No Rights Reserved” license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law, 2021.

Nod factors secreted by nitrogen-fixing rhizobia are lipo-chitooligosaccharidic signals required for establishment of the nodule symbiosis with legumes. In Medicago truncatula, the Nod factor hydrolase 1 (MtNFH1) was found to cleave Nod factors of Sinorhizobium meliloti. Here, we report that the class V chitinase MtCHIT5b of M. truncatula expressed in Escherichia coli can release lipodisaccharides from Nod factors. Analysis of M. truncatula mutant plants indicated that MtCHIT5b, together with MtNFH1, degrades S. meliloti Nod factors in the rhizosphere. MtCHIT5b expression was induced by treatment of roots with purified Nod factors or inoculation with rhizobia. MtCHIT5b with a fluorescent tag was detected in the infection pocket of root hairs. Nodulation of a MtCHIT5b knockout mutant was not significantly altered whereas overexpression of MtCHIT5b resulted in fewer nodules. Reduced nodulation was observed when MtCHIT5b and MtNFH1 were simultaneously silenced in RNA interference experiments. Overall, this study shows that nodule formation of M. truncatula is regulated by a second Nod factor cleaving hydrolase in addition to MtNFH1.

Many plant-encoded E3 ligases are known to be involved in plant defense. Here, we report a novel role of E3 ligase SALT- AND DROUGHT-INDUCED RING FINGER1 (SDIR1) in plant immunity. Even though SDIR1 is reasonably well-characterized, its role in biotic stress response is not known. The silencing of SDIR1 in Nicotiana benthamiana reduced the multiplication of the virulent bacterial pathogen Pseudomonas syringae pv. tabaci. The Arabidopsis sdir1 mutant is resistant to virulent pathogens, whereas SDIR1 overexpression lines are susceptible to both host and nonhost hemibiotrophic bacterial pathogens. However, sdir1 mutant and SDIR1 overexpression lines showed hypersusceptibility and resistance, respectively, against the necrotrophic pathogen Erwinia carotovora. The mutant of SDIR1 target protein, i.e., SDIR-interacting protein 1 (SDIR1P1), also showed resistance to host and nonhost pathogens. In SDIR1 overexpression plants, transcripts of NAC transcription factors were less accumulated and the levels of jasmonic acid (JA) and abscisic acid were increased. In the sdir1 mutant, JA signaling genes JAZ7 and JAZ8 were downregulated. These data suggest that SDIR1 is a susceptibility factor and its activation or overexpression enhances disease caused by P. syringae pv. tomato DC3000 in Arabidopsis. Our results show a novel role of SDIR1 in modulating plant defense gene expression and plant immunity.

Abstract Background Flowering time is an important trait for productivity in legumes, which include many food and fodder plants. Medicago truncatula (Medicago) is a model temperate legume used to study flowering time pathways. Like Arabidopsis thaliana (Arabidopsis), its flowering is promoted by extended periods of cold (vernalization, V), followed by warm long day (LD) photoperiods. However, Arabidopsis flowering-time genes such as the FLOWERING LOCUS C (FLC)/ MADS AFFECTING FLOWERING (MAF) clade are missing and CONSTANS-LIKE (CO-LIKE) genes do not appear to have a role in Medicago or Pisum sativum (pea). Another photoperiodic regulator, the red/far red photoreceptor PHYTOCHROME A (PHYA), promotes Arabidopsis flowering by stabilizing the CO protein in LD. Interestingly, despite the absence of CO-LIKE function in pea, PsPHYA plays a key role in promoting LD photoperiodic flowering and plant architecture. Medicago has one homolog of PHYA, MtPHYA, but its function is not known. Results Genetic analysis of two MtPHYA Tnt1 insertion mutant alleles indicates that MtPHYA has an important role in promoting Medicago flowering and primary stem elongation in VLD and LD and in perception of far-red wavelengths in seedlings. MtPHYA positively regulates the expression of MtE1-like (MtE1L), a homologue of an important legume-specific flowering time gene, E1 in soybean and other Medicago LD-regulated flowering-time gene homologues, including the three FLOWERING LOCUS T-LIKE (FT-LIKE) genes, MtFTa1, MtFTb1 and MtFTb2 and the two FRUITFULL-LIKE (FUL-LIKE) genes MtFULa and MtFULb. MtPHYA also modulates the expression of the circadian clock genes, GIGANTEA (GI) and TIMING OF CAB EXPRESSION 1a (TOC1a). Genetic analyses indicate that Mtphya-1 Mte1l double mutants flowered at the same time as the single mutants. However, Mtphya-1 Mtfta1 double mutants had a weak additive effect in delaying flowering and in reduction of primary axis lengths beyond what was conferred by either of the single mutants. Conclusion MtPHYA has an important role in LD photoperiodic control of flowering, plant architecture and seedling de-etiolation under far-red wavelengths in Medicago. It promotes the expression of LD-induced flowering time genes and modulates clock-related genes. In addition to MtFTa1, MtPHYA likely regulates other targets during LD floral induction in Medicago.

Switchgrass rust caused by Puccinia novopanici (P. novopanici) has the ability to significantly affect the biomass yield of switchgrass, an important biofuel crop in the United States. A comparative genome analysis of P. novopanici with rust pathogen genomes infecting monocot cereal crops wheat, barley, oats, maize and sorghum revealed the presence of larger structural variations contributing to their genome sizes. A comparative alignment of the rust pathogen genomes resulted in the identification of collinear and syntenic relationships between P. novopanici and P. sorghi; P. graminis tritici 21–0 (Pgt 21) and P. graminis tritici Ug99 (Pgt Ug99) and between Pgt 21 and P. triticina (Pt). Repeat element analysis indicated a strong presence of retro elements among different Puccinia genomes, contributing to the genome size variation between ~1 and 3%. A comparative look at the enriched protein families of Puccinia spp. revealed a predominant role of restriction of telomere capping proteins (RTC), disulfide isomerases, polysaccharide deacetylases, glycoside hydrolases, superoxide dismutases and multi-copper oxidases (MCOs). All the proteomes of Puccinia spp. share in common a repertoire of 75 secretory and 24 effector proteins, including glycoside hydrolases cellobiohydrolases, peptidyl-propyl isomerases, polysaccharide deacetylases and protein disulfide-isomerases, that remain central to their pathogenicity. Comparison of the predicted effector proteins from Puccinia spp. genomes to the validated proteins from the Pathogen–Host Interactions database (PHI-base) resulted in the identification of validated effector proteins PgtSR1 (PGTG_09586) from P. graminis and Mlp124478 from Melampsora laricis across all the rust pathogen genomes.

Summary: This protocol describes the analysis of protein cysteine redox status. Redox status is crucial in regulating protein activity, stability, and redox signaling cascades. It is determined by conjugation with 1.24 kDa MM(PEG)24 molecule to each reduced cysteine followed by western blot analysis. This protocol is easy to follow, and most of the reagents and instruments required are of common use in any lab. This protocol can be successfully applied to other biological sources.For complete details on the use and execution of this protocol, please refer to Pant et al. (2020).

Auxin Response Factors (ARFs) constitute a large family of transcription factors that mediate auxin-regulated developmental programs in plants. ARF2, ARF3, and ARF4 are post-transcriptionally regulated by the microRNA390 (miR390)/trans-acting small interference RNA 3 (TAS3) module through the action of TAS3-derived trans-acting small interfering RNAs (ta-siRNA). We have previously reported that constitutive activation of the miR390/TAS3 pathway promotes elongation of lateral roots but impairs nodule organogenesis and infection by rhizobia during the nitrogen-fixing symbiosis established between Medicago truncatula and its partner Sinorhizobium meliloti. However, the involvement of the targets of the miR390/TAS3 pathway, i.e., MtARF2, MtARF3, MtARF4a, and MtARF4b, in root development and establishment of the nitrogen-fixing symbiosis remained unexplored. Here, promoter:reporter fusions showed that expression of both MtARF3 and MtARF4a was associated with lateral root development; however, only the MtARF4a promoter was active in developing nodules. In addition, up-regulation of MtARF2, MtARF3, and MtARF4a/b in response to rhizobia depends on Nod Factor perception. We provide evidence that simultaneous knockdown of MtARF2, MtARF3, MtARF4a, and MtARF4b or mutation in MtARF4a impaired nodule formation, and reduced initiation and progression of infection events. Silencing of MtARF2, MtARF3, MtARF4a, and MtARF4b altered mRNA levels of the early nodulation gene nodulation signaling pathway 2 (MtNSP2). In addition, roots with reduced levels of MtARF2, MtARF3, MtARF4a, and MtARF4b, as well as arf4a mutant plants exhibited altered root architecture, causing a reduction in primary and lateral root length, but increasing lateral root density. Taken together, our results suggest that these ARF members are common key players of the morphogenetic programs that control root development and the formation of nitrogen-fixing nodules.

Flowering time influences the yield and productivity of legume crops. Medicago truncatula is a reference temperate legume that, like the winter annual Arabidopsis thaliana, shows accelerated flowering in response to vernalization (extended cold) and long-day (LD) photoperiods (VLD). However, unlike A. thaliana, M. truncatula appears to lack functional homologs of core flowering time regulators CONSTANS (CO) and FLOWERING LOCUS C (FLC) which act upstream of the mobile florigen FLOWERING LOCUS T (FT). Medicago truncatula has three LD-induced FT-like genes (MtFTa1, MtFTb1, and MtFTb2) with MtFTa1 promoting M. truncatula flowering in response to VLD. Another photoperiodic regulator in A. thaliana, FE, acts to induce FT expression. It also regulates the FT transport pathway and is required for phloem development. Our study identifies a M. truncatula FE homolog Medtr6g444980 (MtFE) which complements the late flowering fe-1 mutant when expressed from the phloem-specific SUCROSE-PROTON SYMPORTER 2 (SUC2) promoter. Analysis of two M. truncatula Tnt1 insertional mutants indicate that MtFE promotes flowering in LD and VLD and growth in all conditions tested. Expression of MtFTa1, MtFTb1, and MtFTb2 are reduced in Mtfe mutant (NF5076), correlating with its delayed flowering. The NF5076 mutant plants are much smaller than wild type indicating that MtFE is important for normal plant growth. The second mutant (NF18291) displays seedling lethality, like strong fe mutants. We searched for mutants in MtFTb1 and MtFTb2 identifying a Mtftb2 knock out Tnt1 mutant (NF20803). However, it did not flower significantly later than wild type. Previously, yeast-two-hybrid assays (Y2H) suggested that Arabidopsis FE interacted with CO and NUCLEAR FACTOR-Y (NF-Y)-like proteins to regulate FT. We found that MtFE interacts with CO and also M. truncatula NF-Y-like proteins in Y2H experiments. Our study indicates that despite the apparent absence of a functional MtCO-like gene, M. truncatula FE likely influences photoperiodic FT expression and flowering time in M. truncatula via a partially conserved mechanism with A. thaliana.

Pathogen associated molecular pattern (PAMP) triggered immunity (PTI) is the first line of plant defense. We hypothesized that the absence of pattern recognition receptors (PRRs) in plants could influence the rhizosphere microbiome. Here, we report sequencing of the 16S ribosomal RNA gene and the fungal ribosomal RNA internal transcribed spacer region of rhizosphere DNA from three Arabidopsis PRR mutants involved in plant innate immunity (efr1, fls2, and cerk1). We conducted experiments in a growth chamber using native soil from the Red River Farm (Terral, OK, USA) to detect microbial community shifts in the rhizosphere that may occur in the absence of PRR receptors compared to wild-type (WT; Col-0) plants. No difference in the α-diversity of the rhizosphere microbial population was observed between the PRR mutants tested and the WT. Plant host genotype had a significant impact in bacterial β-diversity only between the fls2 mutant and the WT. Surprisingly, no significant changes in fungal β-diversity were observed between the PRR mutants and WT, although we observed an increase in relative abundance for the cup fungi (Pezizaceae) in the cerk1 mutant. This finding suggests that the FLS2 receptor can modulate the rhizosphere-associated microbiome β-diversity and expands the list of current known genotypes that can modulate the rhizosphere microbiota.

Summary: Global warming and emerging plant diseases challenge agricultural food/feed production. We identify mechanism(s) regulating both plant thermotolerance and disease resistance. Using virus-induced gene silencing (VIGS)-based genetic screening, we identify a thioredoxin-like 1 (TRXL1) gene involved in plant nonhost disease resistance and thermotolerance. TRXL1 is reduced, partly degraded via proteases and proteasome, and alters its chloroplast localization during heat stress. TRXL1 interacts with more than 400 proteins, including chaperonin CPN60A, caseinolytic protease (CLPC1), and NADP-dependent malate dehydrogenase (NADP-MDH). Chaperonin 60A (CPN60A) guards TRXL1 from degradation, whereas CLPC1 degrades TRXL1 during heat stress. TRXL1 regulates NADP-MDH activity, leading to an increase in malate level and inhibition of superoxide radical formation. We show that CPN60A and NADP-MDH positively regulate nonhost resistance, and CPN60A positively and CLPC1 negatively regulate thermotolerance. This study shows an antagonistic post-translational regulation of TRXL1 by CPN60A and CLPC1 and regulation of MDH by TRXL1, leading to plant disease resistance and thermotolerance.

Abstract l‐Tyrosine (Tyr) is an aromatic amino acid synthesized de novo in plants and microbes downstream of the shikimate pathway. In plants, Tyr and a Tyr pathway intermediate, 4‐hydroxyphenylpyruvate (HPP), are precursors to numerous specialized metabolites, which are crucial for plant and human health. Tyr is synthesized in the plastids by a TyrA family enzyme, arogenate dehydrogenase (ADH/TyrAa), which is feedback inhibited by Tyr. Additionally, many legumes possess prephenate dehydrogenases (PDH/TyrAp), which are insensitive to Tyr and localized to the cytosol. Yet the role of PDH enzymes in legumes is currently unknown. This study isolated and characterized Tnt1‐transposon mutants of MtPDH1 (pdh1) in Medicago truncatula to investigate PDH function. The pdh1 mutants lacked PDH transcript and PDH activity, and displayed little aberrant morphological phenotypes under standard growth conditions, providing genetic evidence that MtPDH1 is responsible for the PDH activity detected in M. truncatula. Though plant PDH enzymes and activity have been specifically found in legumes, nodule number and nitrogenase activity of pdh1 mutants were not significantly reduced compared with wild‐type (Wt) during symbiosis with nitrogen‐fixing bacteria. Although Tyr levels were not significantly different between Wt and mutants under standard conditions, when carbon flux was increased by shikimate precursor feeding, mutants accumulated significantly less Tyr than Wt. These data suggest that MtPDH1 is involved in Tyr biosynthesis when the shikimate pathway is stimulated and possibly linked to unidentified legume‐specific specialized metabolism.

Symbiotic nitrogen fixation carried out by the interaction between legumes and diazotrophic bacteria known as rhizobia requires relatively large levels of transition metals. These elements are cofactors of many key enzymes involved in this process. Metallic micronutrients are obtained from soil by the roots and directed to sink organs by the vasculature, in a process mediated by a number of metal transporters and small organic molecules that facilitate metal delivery in the plant fluids. Among the later, nicotianamine is one of the most important. Synthesized by nicotianamine synthases (NAS), this molecule forms metal complexes participating in intracellular metal homeostasis and long-distance metal trafficking. Here we characterized the NAS2 gene from model legume Medicago truncatula. MtNAS2 is located in the root vasculature and in all nodule tissues in the infection and fixation zones. Symbiotic nitrogen fixation requires of MtNAS2 function, as indicated by the loss of nitrogenase activity in the insertional mutant nas2-1, phenotype reverted by reintroduction of a wild-type copy of MtNAS2. This would result from the altered iron distribution in nas2-1 nodules shown with X-ray fluorescence. Moreover, iron speciation is also affected in these nodules. These data suggest a role of nicotianamine in iron delivery for symbiotic nitrogen fixation.

Abstract Background Plants have evolved excellent ability of flexibly regulating the growth of organs to adapt to changing environment, for example, the modulation of lateral root development in response to environmental stresses. Despite of fundamental discovery that some microRNAs are involved in this process, the molecular mechanisms of how these microRNAs work together are still largely unknown. Results Here we show that miR390 induced by auxin promotes lateral root growth in rice. However, this promotion can be suppressed by miR393, which is induced by various stresses and ABA (Abscisic Acid). Results that miR393 responded to ABA stronger and earlier than other stresses implied that ABA likely is authentic factor for inducing miR393. The transgenic lines respectively over-expressing miR393 and OsTAS3a (Oryza sativa Trans-Acting Short RNA precursor 3a) displayed opposite phenotypes in lateral root growth. MiR390 was found to be dominantly expressed at lateral root primordia and roots tips while miR393 mainly expressed in the base part of roots at very low level. When miR393 was up-regulated by various stresses, miR390 expression level fell down. However, the risen expression level of miR390 induced by auxin didn’t affect the expression of miR393 and its target OsTIR1 (Transport Inhibitor Response 1). Together with analysis of the two transgenic lines, we provide a model of how the growth of lateral roots in rice is regulated distinctively by the 2 microRNAs. Conclusion We propose that miR390 induced by auxin triggers the lateral root growth under normal growth conditions, meanwhile miR393 just lurks as a potentially regulative role; Once plants suffer from stresses, miR393 will be induced to negatively regulate miR390-mediated growth of lateral roots in rice.

Abstract Background Although the draft genome of sorghum is available, the understanding of gene function is limited due to the lack of extensive mutant resources. Virus-induced gene silencing (VIGS) is an alternative to mutant resources to study gene function. This study reports an improved and efficient method for Brome mosaic virus (BMV)-based VIGS in sorghum. Methods Sorghum plants were rub-inoculated with sap prepared by grinding 2 g of infected Nicotiana benthamiana leaf in 1 ml 10 mM potassium phosphate buffer (pH 6.8) and 100 mg of carborundum abrasive. The sap was rubbed on two to three top leaves of sorghum. Inoculated plants were covered with a dome to maintain high humidity and kept in the dark for two days at 18 °C. Inoculated plants were then transferred to 18 °C growth chamber with 12 h/12 h light/dark cycle. Results This study shows that BMV infection rate can be significantly increased in sorghum by incubating plants at 18 °C. A substantial variation in BMV infection rate in sorghum genotypes/varieties was observed and BTx623 was the most susceptible. Ubiquitin (Ubiq) silencing is a better visual marker for VIGS in sorghum compared to other markers such as Magnesium Chelatase subunit H (ChlH) and Phytoene desaturase (PDS). The use of antisense strand of a gene in BMV was found to significantly increase the efficiency and extent of VIGS in sorghum. In situ hybridization experiments showed that the non-uniform silencing in sorghum is due to the uneven spread of the virus. This study further demonstrates that genes could also be silenced in the inflorescence of sorghum. Conclusion In general, sorghum plants are difficult to infect with BMV and therefore recalcitrant to VIGS studies. However, by using BMV as a vector, a BMV susceptible sorghum variety, 18 °C for incubating plants, and antisense strand of the target gene fragment, efficient VIGS can still be achieved in sorghum.

Chloroplasts have a crucial role in plant immunity against pathogens. Increasing evidence suggests that phytopathogens target chloroplast homeostasis as a pathogenicity mechanism. In order to regulate the performance of chloroplasts under stress conditions, chloroplasts produce retrograde signals to alter nuclear gene expression. Many signals for the chloroplast retrograde pathway have been identified, including chlorophyll intermediates, reactive oxygen species, and metabolic retrograde signals. Although there is a reasonably good understanding of chloroplast retrograde signaling in plant immunity, some signals are not well-understood. In order to understand the role of chloroplast retrograde signaling in plant immunity, we investigated Arabidopsis chloroplast retrograde signaling mutants in response to pathogen inoculation. sal1 mutants (fry1-2 and alx8) responsible for the SAL1-PAP retrograde signaling pathway showed enhanced disease symptoms not only to the hemibiotrophic pathogen Pseudomonas syringae pv. tomato DC3000 but, also, to the necrotrophic pathogen Pectobacterium carotovorum subsp. carotovorum EC1. Glucosinolate profiles demonstrated the reduced accumulation of aliphatic glucosinolates in the fry1-2 and alx8 mutants compared with the wild-type Col-0 in response to DC3000 infection. In addition, quantification of multiple phytohormones and analyses of their gene expression profiles revealed that both the salicylic acid (SA)- and jasmonic acid (JA)-mediated signaling pathways were down-regulated in the fry1-2 and alx8 mutants. These results suggest that the SAL1-PAP chloroplast retrograde pathway is involved in plant immunity by regulating the SA- and JA-mediated signaling pathways.

Abstract Drought transcriptome analysis of finger millet (Eleusine coracana) by cDNA subtraction identified drought responsive genes that have a potential role in drought tolerance. Through virus-induced gene silencing (VIGS) in a related crop species, maize (Zea mays), several genes, including a G-BOX BINDING FACTOR 3 (GBF3) were identified as candidate drought stress response genes and the role of GBF3 in drought tolerance was studied in Arabidopsis thaliana. Overexpression of both EcGBF3 and AtGBF3 in A. thaliana resulted in improved tolerance to osmotic stress, salinity and drought stress in addition to conferring insensitivity to ABA. Conversely, loss of function of this gene increased the sensitivity of A. thaliana plants to drought stress. EcGBF3 transgenic A. thaliana results also suggest that drought tolerance of sensitive plants can be improved by transferring genes from far related crops like finger millet. Our results demonstrate the role of GBF3 in imparting drought tolerance in A. thaliana and indicate the conserved role of this gene in drought and other abiotic stress tolerance in several plant species.

Abstract Plant defense responses at stomata and apoplast are the most important early events during plant-bacteria interactions. The key components for the signaling of stomatal defense and nonhost resistance have not been fully characterized. Here we report the newly identified small GTPase, Nucleolar GTP-binding protein 1 (NOG1), functions for plant immunity against bacterial pathogens. Virus-induced gene silencing of NOG1 compromised nonhost resistance in N. benthamiana and tomato. Comparative genomic analysis showed that two NOG1 copies are present in all known plant species: NOG1-1 and NOG1-2. Gene downregulation and overexpression studies of NOG1-1 and NOG1-2 in Arabidopsis revealed the novel function of these genes in nonhost resistance and stomatal defense against bacterial pathogens, respectively. Specially, NOG1-2 regulates guard cell signaling in response to biotic and abiotic stimuli through jasmonic acid (JA)- and abscisic acid (ABA)-mediated pathways. The results here provide valuable information on the new functional role of small GTPase, NOG1, in guard cell signaling and early plant defense in response to bacterial pathogens.

The constant interactions between plants and pathogens in the environment and the resulting outcomes are of significant importance for agriculture and agricultural scientists. Disease resistance genes in plant cultivars can break down in the field due to the evolution of pathogens under high selection pressure. Thus, the protection of crop plants against pathogens is a continuous arms race. Like any other type of crop plant, legumes are susceptible to many pathogens. The dawn of the genomic era, in which high-throughput and cost-effective genomic tools have become available, has revolutionized our understanding of the complex interactions between legumes and pathogens. Genomic tools have enabled a global view of transcriptome changes during these interactions, from which several key players in both the resistant and susceptible interactions have been identified. This review summarizes some of the large-scale genomic studies that have clarified the host transcriptional changes during interactions between legumes and their plant pathogens while highlighting some of the molecular breeding tools that are available to introgress the traits into breeding programs. These studies provide valuable insights into the molecular basis of different levels of host defenses in resistant and susceptible interactions.

Optimizing flowering time is crucial for maximizing crop productivity, but gaps remain in the knowledge of the mechanisms underpinning temperate legume flowering. Medicago, like winter annual Arabidopsis, accelerates flowering after exposure to extended cold (vernalization, V) followed by long-day (LD) photoperiods. In Arabidopsis, photoperiodic flowering is triggered through CO, a photoperiodic switch that directly activates the FT gene encoding a mobile florigen and potent activator of flowering. In Arabidopsis, several CYCLING DOF FACTORs (CDFs), including AtCDF1, act redundantly to repress CO and thus FT expression, until their removal in LD by a blue-light-induced F-BOX1/GIGANTEA (FKF1/GI) complex. Medicago possesses a homolog of FT, MtFTa1, which acts as a strong activator of flowering. However, the regulation of MtFTa1 does not appear to involve a CO-like gene. Nevertheless, work in pea suggests that CDFs may still regulate flowering time in temperate legumes. Here, we analyze the function of Medicago MtCDF genes with a focus on MtCDFd1_1 in flowering time and development. MtCDFd1_1 causes strong delays to flowering when overexpressed in Arabidopsis and shows a cyclical diurnal expression in Medicago with peak expression at dawn, consistent with AtCDF genes like AtCDF1. However, MtCDFd1_1 lacks predicted GI or FKF1 binding domains, indicating possible differences in its regulation from AtCDF1. In Arabidopsis, CDFs act in a redundant manner, and the same is likely true of temperate legumes as no flowering time phenotypes were observed when MtCDFd1_1 or other MtCDFs were knocked out in Medicago Tnt1 lines. Nevertheless, overexpression of MtCDFd1_1 in Medicago plants resulted in late flowering relative to wild type in inductive vernalized long-day (VLD) conditions, but not in vernalized short days (VSDs), rendering them day neutral. Expression of MtCO-like genes was not affected in the transgenic lines, but LD-induced genes MtFTa1, MtFTb1, MtFTb2, and MtSOC1a showed reduced expression. Plants carrying both the Mtfta1 mutation and 35S:MtCDFd1_1 flowered no later than the Mtfta1 plants. This indicates that 35S:MtCDFd1_1 likely influences flowering in VLD via repressive effects on MtFTa1 expression. Overall, our study implicates MtCDF genes in photoperiodic regulation in Medicago by working redundantly to repress FT-like genes, particularly MtFTa1, but in a CO-independent manner, indicating differences from the Arabidopsis model.

Gene expression data generated from multiple biological samples (mutant, double mutant, and wild-type) are often compared via Venn diagram tools. It is of great interest to know the expression pattern between overlapping genes and their associated gene pathways or gene ontology (GO) terms. We developed DiVenn (Dive into the Venn diagram and create a force directed graph)—a novel web-based tool that compares gene lists from multiple RNA-Seq experiments in a force-directed graph, which shows the gene regulation levels for each gene and integrated KEGG pathway and gene ontology knowledge for the data visualization. DiVenn has four key features: (1) informative force-directed graph with gene expression levels to compare multiple data sets; (2) interactive visualization with biological annotations and integrated pathway and GO databases, which can be used to subset or highlight gene nodes to pathway or GO terms of interest in the graph; (3) Pathway and GO enrichment analysis of all or selected genes in the graph; and (4) high resolution image and gene-associated information export. DiVenn is freely available at http://divenn.noble.org/.

Bacterial Nod Factors (NF) regulate the formation of nitrogen fixing symbiotic nodules on legume roots. Here, the authors show that gibberellins act via DELLA proteins to regulate nodulation and propose that DELLA acts with NSP2 and NF-YA1 as a transcriptional co-activator of ERN1-dependent NF signalling.

The C function gene AGAMOUS (AG) encodes for a MADS-box transcription factor required for floral organ identity and floral meristem (FM) determinacy in angiosperms. Unlike Arabidopsis, most legume plants possess two AG homologs arose by an ancient genome duplication event. Recently, two euAGAMOUS genes, MtAGa and MtAGb, were characterized and shown to fulfill the C function activity in the model legume Medicago truncatula. Here, we reported the isolation and characterization of a new mtaga allele by screening the Medicago Tnt1 insertion mutant collection. We found that MtAGa was not only required for controlling the stamen and carpel identity but also affected pod and seed development. Genetic analysis indicated that MtAGa and MtAGb redundantly control Medicago floral organ identity, but have minimal distinct functions in regulating stamen and carpel development in a dose-dependent manner. Interestingly, the stamens and carpels are mostly converted to numerous vexillum-like petals in the double mutant of mtaga mtagb, which is distinguished from Arabidopsis ag. Further qRT-PCR analysis in different mtag mutants revealed that MtAGa and MtAGb can repress the expression of putative A and B function genes as well as MtWUS, but promote putative D function genes expression in M. truncatula. In addition, we found that the abnormal dorsal petal phenotype observed in the mtaga mtagb double mutant is associated with the upregulation of CYCLOIDEA (CYC)-like TCP genes. Taken together, our data suggest that the redundant MtAGa and MtAGb genes of M. truncatula employ a conserved mechanism of action similar to Arabidopsis in determining floral organ identity and FM determinacy but may have evolved distinct function in regulating floral symmetry by coordinating with specific floral dorsoventral identity factors.

Abstract Virus‐induced gene silencing (VIGS) is an important forward and reverse genetics method for the study of gene function in many plant species, especially Nicotiana benthamiana. However, despite the widespread use of VIGS, a searchable database compiling the phenotypes observed with this method is lacking. Such a database would allow researchers to know the phenotype associated with the silencing of a large number of individual genes without experimentation. We have developed a VIGS phenomics and functional genomics database (VPGD) that has DNA sequence information derived from over 4,000 N. benthamiana VIGS clones along with the associated silencing phenotype for approximately 1,300 genes. The VPGD has a built‐in BLAST search feature that provides silencing phenotype information of specific genes. In addition, a keyword‐based search function could be used to find a specific phenotype of interest with the corresponding gene, including its Gene Ontology descriptions. Query gene sequences from other plant species that have not been used for VIGS can also be searched for their homologs and silencing phenotype in N. benthamiana. VPGD is useful for identifying gene function not only in N. benthamiana but also in related Solanaceae plants such as tomato and potato. The database is accessible at http://vigs.noble.org.

Like Arabidopsis thaliana, the flowering of the legume Medicago truncatula is promoted by long day (LD) photoperiod and vernalization. However, there are differences in the molecular mechanisms involved, with orthologs of two key Arabidopsis thaliana regulators, FLOWERING LOCUS C (FLC) and CONSTANS (CO), being absent or not having a role in flowering time function in Medicago. In Arabidopsis, the MADS-box transcription factor gene, SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (AtSOC1), plays a key role in integrating the photoperiodic and vernalization pathways. In this study, we set out to investigate whether the Medicago SOC1 genes play a role in regulating flowering time. Three Medicago SOC1 genes were identified and characterized (MtSOC1a–MtSOC1c). All three MtSOC1 genes, when heterologously expressed, were able to promote earlier flowering of the late-flowering Arabidopsis soc1-2 mutant. The three MtSOC1 genes have different patterns of expression. However, consistent with a potential role in flowering time regulation, all three MtSOC1 genes are expressed in the shoot apex and are up-regulated in the shoot apex of plants in response to LD photoperiods and vernalization. The up-regulation of MtSOC1 genes was reduced in Medicago fta1-1 mutants, indicating that they are downstream of MtFTa1. Insertion mutant alleles of Medicago soc1b do not flower late, suggestive of functional redundancy among Medicago SOC1 genes in promoting flowering.

Legume plants form symbiotic associations with either nitrogen-fixing bacteria or arbuscular mycorrhizal (AM) fungi, which are regulated by a set of common symbiotic signaling pathway genes. Central to the signaling pathway is the activation of the DMI3/IPD3 protein complex by Ca2+ oscillations, and the initiation of nodule organogenesis and mycorrhizal symbiosis. DMI3 is essential for rhizobial infection and nodule organogenesis; however, ipd3 mutants have been shown to be impaired only in infection thread formation but not in root nodule organogenesis in Medicago truncatula. We identified an IPD3-like (IPD3L) gene in the M. truncatula genome. A single ipd3l mutant exhibits a normal root nodule phenotype. The ipd3l/ipd3-2 double mutant is completely unable to initiate infection threads and nodule primordia. IPD3L can functionally replace IPD3 when expressed under the control of the IPD3 promoter, indicating functional redundancy between these two transcriptional regulators. We constructed a version of IPD3 that was phosphomimetic with respect to two conserved serine residues (IPD3-2D). This was sufficient to trigger root nodule organogenesis, but the increased multisite phosphorylation of IPD3 (IPD3-8D) led to low transcriptional activity, suggesting that the phosphorylation levels of IPD3 fine-tune its transcriptional activity in the root nodule symbiosis. Intriguingly, the phosphomimetic version of IPD3 triggers spontaneous root-like nodules on the roots of dmi3-1 and dmi2-1 (DMI2 is an LRR-containing receptor-like kinase gene which is required for Ca2+ spiking), but not on the roots of wild-type or ipd3l ipd3-2 plants. In addition, fully developed arbuscules were formed in the ipd3l ipd3-2 mutants but not the ccamk/dmi3-1 mutants. Collectively, our data indicate that, in addition to IPD3 and IPD3L, another new genetic component or other new phosphorylation sites of IPD3 function downstream of DMI3 in rhizobial and mycorrhizal symbioses.

Chloroplasts are cytoplasmic organelles for photosynthesis in eukaryotic cells. In addition, recent studies have shown that chloroplasts have a critical role in plant innate immunity against invading pathogens. Hydrogen peroxide is a toxic by-product from photosynthesis, which also functions as a signaling compound in plant innate immunity. Therefore, it is important to regulate the level of hydrogen peroxide in response to pathogens. Chloroplasts maintain components of the redox detoxification system including enzymes such as 2-Cys peroxiredoxins (2-Cys Prxs), and NADPH-dependent thioredoxin reductase C (NTRC). However, the significance of 2-Cys Prxs and NTRC in the molecular basis of nonhost disease resistance is largely unknown. We evaluated the roles of Prxs and NTRC using knock-out mutants of Arabidopsis in response to nonhost Pseudomonas syringae pathogens. Plants lacking functional NTRC showed localized cell death (LCD) accompanied by the elevated accumulation of hydrogen peroxide in response to nonhost pathogens. Interestingly, the Arabidopsis ntrc mutant showed enhanced bacterial growth and disease susceptibility of nonhost pathogens. Furthermore, the expression profiles of the salicylic acid (SA) and jasmonic acid (JA)-mediated signaling pathways and phytohormone analyses including SA and JA revealed that the Arabidopsis ntrc mutant shows elevated JA-mediated signaling pathways in response to nonhost pathogen. These results suggest the critical role of NTRC in plant innate immunity against nonhost P. syringae pathogens.

Coronatine (COR)-producing pathovars of Pseudomonas syringae, including pvs. tomato, maculicola, and glycinea, cause important diseases on tomato, crucifers, and soybean, respectively, and produce symptoms with necrotic lesions surrounded by chlorosis. The chlorosis is mainly attributed to COR. However, the significance of COR-induced chlorosis in localized lesion development and the molecular basis of disease-associated cell death is largely unknown. To identify host (chloroplast) genes that play a role in COR-mediated chlorosis, we used a forward genetics approach using Nicotiana benthamiana and virus-induced gene silencing and identified a gene which encodes 2-Cys peroxiredoxin (Prxs) that, when silenced, produced a spreading hypersensitive or necrosis-like phenotype instead of chlorosis after COR application in a COI1-dependent manner. Loss-of-function analysis of Prx and NADPH-dependent thioredoxin reductase C (NTRC), the central players of a chloroplast redox detoxification system, resulted in spreading accelerated P. syringae pv. tomato DC3000 disease-associated cell death with enhanced reactive oxygen species (ROS) accumulation in a COR-dependent manner in tomato and Arabidopsis. Consistent with these results, virulent strain DC3000 suppressed the expression of Prx and NTRC in Arabidopsis and tomato during pathogenesis. However, interestingly, authentic COR suppressed the expression of Prx and NTRC in tomato but not in Arabidopsis, suggesting that COR in conjunction with other effectors may modulate ROS and cell death in different host species. Taken together, these results indicated that NTRC or Prx function as a negative regulator of pathogen-induced cell death in the healthy tissues that surround the lesions, and COR-induced chloroplast-localized ROS play a role in enhancing the disease-associated cell death.

Legumes form endosymbiotic associations with nitrogen-fixing bacteria and arbuscular mycorrhizal (AM) fungi which facilitate nutrient uptake. Both symbiotic interactions require a molecular signal exchange between the plant and the symbiont, and this involves a conserved symbiosis (Sym) signaling pathway. In order to identify plant genes required for intracellular accommodation of nitrogen-fixing bacteria and AM fungi, we characterized Medicago truncatula symbiotic mutants defective for rhizobial infection of nodule cells and colonization of root cells by AM hyphae. Here, we describe mutants impaired in the interacting protein of DMI3 (IPD3) gene, which has been identified earlier as an interacting partner of the calcium/calmodulin-dependent protein, a member of the Sym pathway. The ipd3 mutants are impaired in both rhizobial and mycorrhizal colonization and we show that IPD3 is necessary for appropriate Nod-factor-induced gene expression. This indicates that IPD3 is a member of the common Sym pathway. We observed differences in the severity of ipd3 mutants that appear to be the result of the genetic background. This supports the hypothesis that IPD3 function is partially redundant and, thus, additional genetic components must exist that have analogous functions to IPD3. This explains why mutations in an essential component of the Sym pathway have defects at late stages of the symbiotic interactions.

Phymatotrichopsis omnivora (Duggar) Hennebert causes a destructive root rot in cotton, alfalfa (Medicago sativa), and many other dicot species. No consistently effective control measures or resistant host germplasm for Phymatotrichum root rot (PRR) are known. The relative genetic intractability of cotton and alfalfa precludes their use as model pathosystem hosts for P. omnivora. Therefore, we used the model legume M. truncatula and its available genetic and genomic resources to investigate PRR. Confocal imaging of P. omnivora interactions with M. truncatula roots revealed that the mycelia do not form any specialized structures for penetration and mainly colonize cortical cells and, eventually, form a mycelial mantle covering the root's surfaces. Expression profiling of M. truncatula roots infected by P. omnivora identified several upregulated genes, including the pathogenesis-related class I and class IV chitinases and genes involved in reactive oxygen species generation and phytohormone (jasmonic acid and ethylene) signaling. Genes involved in flavonoid biosynthesis were induced (2.5- to 10-fold over mock-inoculated controls) at 3 days postinoculation (dpi) in response to fungal penetration. However, the expression levels of flavonoid biosynthesis genes returned to the basal levels with the progress of the disease at 5 dpi. These transcriptome results, confirmed by real-time quantitative polymerase chain reaction analyses, showed that P. omnivora apparently evades induced host defenses and may downregulate phytochemical defenses at later stages of infection to favor pathogenesis.

Bacterial speck disease, which is caused by Pseudomonas syringae pv. tomato, is an economically important disease on tomato. In the present study, we show that P. syringae pv. tomato DC3000 is a pathogen of tomato seedlings, an aspect of pathogen biology that has not been previously investigated. This resulted in the development of a virulence assay on tomato seedlings that has several advantages over labor-intensive foliar assays, including a shorter growth and incubation period, ease of inoculation and handling, and rapid generation of larger sample sizes per experiment. The utility of this assay was investigated by exploring the virulence function of coronatine (COR) on tomato seedlings. Using the COR– mutant DB29 and a MAPMAN display of transcript data from TOM1 microarrays, COR-dependent expression of genes involved in secondary metabolism, polyamine biosynthesis, reactive oxygen species homeostasis, and the novel transcription factor SlNAC2 were identified. Furthermore, during pathogenesis, genes involved in photosynthetic light reactions and the Calvin-Benson cycle were strongly repressed by COR. In conclusion, we show that P. syringae pv. tomato infects tomato seedlings and that COR is required for virulence in seedlings. The seedling assay can be used in high-throughput screens for the identification of molecular targets for COR and for the identification of genes involved in pathogenesis.

The roles of the phytotoxin coronatine (COR) and salicylic acid (SA)-mediated defenses in the interaction of Pseudomonas syringae pv. tomato DC3000 and tomato (Solanum lycopersicum) were investigated. Unlike findings reported for Arabidopsis thaliana, DC3000 mutants impaired for production of COR or one of its components, coronafacic acid (CFA) or coronamic acid (CMA), induced distinctly different disease lesion phenotypes in tomato. Tomato plants inoculated with the CFA- CMA- mutant DB29 showed elevated transcript levels of SlICS, which encodes isochorismate synthase, an enzyme involved in SA biosynthesis in S. lycopersicum. Furthermore, expression of genes encoding SA-mediated defense proteins were elevated in DB29-inoculated plants compared with plants inoculated with DC3000, suggesting that COR suppresses SlICS-mediated SA responses. Sequence analysis of SlICS revealed that it encodes a protein that is 55 and 59.6% identical to the A. thaliana ICS-encoded proteins AtICS1 and AtICS2, respectively. Tomato plants silenced for SlICS were hypersusceptible to DC3000 and accumulated lower levels of SA after infection with DC3000 compared with inoculated wild-type tomato plants. Unlike what has been shown for A. thaliana, the COR- mutant DB29 was impaired for persistence in SlICS-silenced tomato plants; thus, COR has additional roles in virulence that are SA independent and important in the latter stages of disease development. In summary, the infection assays, metabolic profiling, and gene expression results described in this study indicate that the intact COR molecule is required for both suppression of SA-mediated defense responses and full disease symptom development in tomato.

Genetic transformation of plant cells by Agrobacterium tu-mefaciens represents a unique case of trans-kingdom sex requiring the involvement of both bacterial virulence proteins and plant-encoded proteins. We have developed in planta and leaf-disk assays in Nicotiana benthamiana for identifying plant genes involved in Agrobacterium-mediated plant transformation using virus-induced gene silencing (VIGS) as a genomics tool. VIGS was used to validate the role of several genes that are either known or speculated to be involved in Agrobacterium-mediated plant transformation. We showed the involvement of a nodulin-like protein and an alpha-expansin protein (α-Exp) during Agrobacterium infection. Our data suggest that α-Exp is involved during early events of Agrobacterium-mediated transformation but not required for attaching A. tumefaciens. By employing the combination of the VIGS-mediated forward genetics approach and an in planta tumorigenesis assay, we identified 21 ACG (altered crown gall) genes that, when silenced, produced altered crown gall phenotypes upon infection with a tumorigenic strain of A. tumefaciens. One of the plant genes identified from the screening, Histone H3 (H3), was further characterized for its biological role in Agrobacterium-mediated plant transformation. We provide evidence for the role of H3 in transfer DNA integration. The data presented here suggest that the VIGS-based approach to identify and characterize plant genes involved in genetic transformation of plant cells by A. tumefaciens is simple, rapid, and robust and complements other currently used approaches.

Switchgrass rust caused by is a major limiting factor for switchgrass ( L.) production, especially in monoculture. Natural populations of switchgrass displayed diverse reactions to when evaluated in an Ardmore, OK, field. To identify the differentially expressed genes during the rust infection process and the mechanisms of switchgrass rust resistance, transcriptome analysis using RNA-Seq was conducted in two pseudo-F parents (‘PV281’ and ‘NFGA472’), and three moderately resistant and three susceptible progenies selected from a three-generation, four-founder switchgrass population (K5 × A4) × (AP13 × VS16). On average, 23.5 million reads per sample (leaf tissue was collected at 0, 24, and 60 h post-inoculation (hpi)) were obtained from paired-end (2 × 100 bp) sequencing on the Illumina HiSeq2000 platform. Mapping of the RNA-Seq reads to the switchgrass reference genome (AP13 ver. 1.1 assembly) constructed a total of 84,209 transcripts from 98,007 gene loci among all of the samples. Further analysis revealed that host defense-related genes, including the nucleotide binding site–leucine-rich repeat domain containing disease resistance gene analogs, play an important role in resistance to rust infection. Rust-induced gene (RIG) transcripts inherited across generations were identified. The rust-resistant gene transcripts can be a valuable resource for developing molecular markers for rust resistance. Furthermore, the rust-resistant genotypes and gene transcripts identified in this study can expedite rust-resistant cultivar development in switchgrass.

Many gram-negative bacterial pathogens rely on a type III secretion system to deliver a number of effector proteins into the host cell. Though a number of these effectors have been shown to contribute to bacterial pathogenicity, their functions remain elusive. Here we report that AvrPto, an effector known for its ability to interact with Pto and induce Pto-mediated disease resistance, inhibited the hypersensitive response (HR) induced by nonhost pathogen interactions. Pseudomonas syringae pv. tomato T1 causes an HR-like cell death on Nicotiana benthamiana. This rapid cell death was delayed significantly in plants inoculated with P. syringae pv. tomato expressing avrPto. In addition, P. syringae pv. tabaci expressing avrPto suppressed nonhost HR on tomato prf3 and ptoS lines. Transient expression of avrPto in both N. benthamiana and tomato prf3 plants also was able to suppress nonhost HR. Interestingly, AvrPto failed to suppress cell death caused by other elicitors and nonhost pathogens. AvrPto also failed to suppress cell death caused by certain gene-for-gene disease resistance interactions. Experiments with avrPto mutants revealed several residues important for the suppression effects. AvrPto mutants G2A, G99V, P146L, and a 12-amino-acid C-terminal deletion mutant partially lost the suppression ability, whereas S94P and I96T enhanced suppression of cell death in N. benthamiana. These results, together with other discoveries, demonstrated that suppression of host-programmed cell death may serve as one of the strategies bacterial pathoens use for successful invasion.

A transgenic wheat line constitutively expressing genes encoding a class IV acidic chitinase and an acidic β-1,3-glucanase, showed significant delay in spread of Fusarium head blight (scab) disease under greenhouse conditions. In an earlier work, we observed a lesion-mimic phenotype in this transgenic line when homozygous for transgene loci. Apoplastic fluid (AF) extracted from the lesion-mimic plants had pathogenesis-related (PR) proteins belonging to families of β-1,3-glucanases, chitinases, and thaumatin-like proteins (TLPs). AF had growth inhibitory activity against certain fungal pathogens, including Fusarium graminearum and Gaeumannomyces graminis var. tritici. Through a two-step ion-exchange chromatography protocol, we recovered many PR proteins and a few uncharacterized proteins. Three individual protein bands corresponding to a TLP (molecular mass, 16 kDa) and two β-1,3-glucanases (molecular mass, 32 kDa each) were purified and identified by tandem mass spectrometry. We measured the in vitro antifungal activity of the three purified enzymes and a barley class II chitinase (purified earlier in our laboratory) in microtiter plate assays with macroconidia or conidiophores of F. graminearum and Pyrenophora tritici-repentis. Mixtures of proteins revealed synergistic or additive inhibitory activity against F. graminearum and P. tritici-repentis hyphae. The concentrations of PR proteins at which these effects were observed are likely to be those reached in AF of cells exhibiting a hypersensitive response. Our results suggest that apoplastic PR proteins are antifungal and their antimicrobial potency is dependent on concentrations and combinations that are effectively reached in plants following microbial attack.

The Arabidopsis thaliana mutants uvh1 and rad5, originally identified as radiation hypersensitive, were reported to be deficient in T-DNA integration based on the relative efficiencies of stable transformation and T-DNA transfer. We reassessed these mutants for susceptibility to transformation by Agrobacterium tumefaciens. The mutant rad5 showed a significant reduction in the efficiency of transient as well as stable transformation, compared with its wild-type progenitor. These data indicate that rad5 is blocked at a step in the transformation process prior to T-DNA integration. We additionally found, using both an in vitro root inoculation and an in vivo flower bolt inoculation assay, that the mutant uvh1 is as susceptible to A. tumefaciens-mediated transformation as is its wild-type progenitor, C10.

VirD2 is one of the key Agrobacterium tumefaciens proteins involved in T-DNA processing and transfer. In addition to its endonuclease domain, VirD2 contains a bipartite C-terminal nuclear localization sequence (NLS) and a conserved region called ω that is important for virulence. Previous results from our laboratory indicated that the C-terminal, bipartite NLS and the ω region are not essential for nuclear uptake of T-DNA, and further suggested that the ω domain may be required for efficient integration of T-DNA into the plant genome. In this study, we took two approaches to investigate the importance of the ω domain in T-DNA integration. Using the first approach, we constructed a T-DNA binary vector containing a promoterless gusA-intron gene just inside the right T-DNA border. The expression of β-glucuronidase (GUS) activity in plant cells transformed by this T-DNA would indicate that the T-DNA integrated downstream of a plant promoter. Approximately 0.4% of the tobacco cell clusters infected by a wild-type A. tumefaciens strain harboring this vector stained blue with 5-bromo-4-chloro-3-indolyl β-d-glucuronic acid (X-gluc). However, using an ω-mutant A. tumefaciens strain harboring the same binary vector, we did not detect any blue staining. Using the second approach, we directly demonstrated that more T-DNA is integrated into high-molecular-weight plant DNA after infection of Arabidopsis thaliana cells with a wild-type A. tumefaciens strain than with a strain containing a VirD2 ω deletion/substitution. Taken together, these data indicate that the VirD2 ω domain is important for efficient T-DNA integration. To determine whether the use of the T-DNA right border is altered in those few tumors generated by A. tumefaciens strains harboring the ω mutation, we analyzed DNA extracted from these tumors. Our data indicate that the right border was used to integrate the T-DNA in a similar manner regardless of whether the VirD2 protein encoded by the inciting A. tumefaciens was wild-type or contained an ω mutation. In addition, a mutant VirD2 protein lacking the ω domain was as least as active in cleaving a T-DNA border in vitro as was the wild-type protein. Finally, we investigated the role of various amino acids of the ω and bipartite NLS domains in the targeting of a GUS-VirD2 fusion protein to the nucleus of electroporated tobacco protoplasts. Deletion of the ω domain, or mutation of the 10-amino-acid region between the two components of the bipartite NLS, had little effect upon the nuclear targeting of the GUS-VirD2 fusion protein. Mutation of both components of the NLS reduced, but did not eliminate, targeting of the fusion protein to the nucleus.

Pseudomonas syringae is the most widespread bacterial pathogen in plants. Several strains of P. syringae produce a phytotoxin, coronatine (COR), which acts as a jasmonic acid mimic and inhibits plant defense responses and contributes to disease symptom development. In this study, we found that COR inhibits early defense responses during nonhost disease resistance. Stomatal closure induced by a nonhost pathogen, P. syringae pv. tabaci, was disrupted by COR in tomato epidermal peels. In addition, nonhost HR cell death triggered by P. syringae pv. tabaci on tomato was remarkably delayed when COR was supplemented along with P. syringae pv. tabaci inoculation. Using isochorismate synthase (ICS)-silenced tomato plants and transcript profiles of genes in SA- and JA-related defense pathways, we show that COR suppresses SA-mediated defense during nonhost resistance.

Nonhost disease resistance is the most common type of plant defense mechanism against potential pathogens. In the present study, the metabolic enzyme formate dehydrogenase 1 (FDH1) was identified to associate with nonhost disease resistance in Nicotiana benthamiana and Arabidopsis thaliana. In Arabidopsis, AtFDH1 was highly upregulated in response to both host and nonhost bacterial pathogens. The Atfdh1 mutants were compromised in nonhost resistance, basal resistance, and gene-for-gene resistance. The expression patterns of salicylic acid (SA) and jasmonic acid (JA) marker genes after pathogen infections in Atfdh1 mutant indicated that both SA and JA are involved in the FDH1-mediated plant defense response to both host and nonhost bacterial pathogens. Previous studies reported that FDH1 localizes to mitochondria, or both mitochondria and chloroplasts. Our results showed that the AtFDH1 mainly localized to mitochondria, and the expression level of FDH1 was drastically increased upon infection with host or nonhost pathogens. Furthermore, we identified the potential co-localization of mitochondria expressing FDH1 with chloroplasts after the infection with nonhost pathogens in Arabidopsis. This finding suggests the possible role of FDH1 in mitochondria and chloroplasts during defense responses against bacterial pathogens in plants.