Soybean-Phakopsora pachyrhizi interactions: towards the development of next-generation disease-resistant plants.

Soybean rust (SBR), caused by the obligate biotrophic fungus Phakopsora pachyrhizi, is a devastating foliar disease threatening soybean production. To date, no commercial cultivars conferring durable resistance to SBR are available. The development of long-lasting SBR resistance has been hindered by the lack of understanding of this complex pathosystem, encompassing challenges posed by intricate genetic structures in both the host and pathogen, leading to a gap in the knowledge of gene-for-gene interactions between soybean and P. pachyrhizi. In this review, we focus on recent advancements and emerging technologies that can be used to improve our understanding of the P. pachyrhizi-soybean molecular interactions. We further explore approaches used to combat SBR, including conventional breeding, transgenic approaches and RNA interference, and how advances in our understanding of plant immune networks, the availability of new molecular tools, and the recent sequencing of the P. pachyrhizi genome could be used to aid in the development of better genetic resistance against SBR. Lastly, we discuss the research gaps of this pathosystem and how new technologies can be used to shed light on these questions and to develop durable next-generation SBR-resistant soybean plants.

Mapping of a soybean rust resistance in PI 594756 at the Rpp1 locus.

Asian soybean rust (ASR), caused by the fungus Phakopsora pachyrhizi, is the main disease affecting soybean in Brazil. This study aimed at investigating and mapping the resistance of the PI 594756 to P. pachyrhizi, by using Bulked Segregant Analysis (BSA). The PI 594756 and the susceptible PI 594891 were crossed and the resulting F2 and F2:3 populations (208 and 1770 plants, respectively) were tested against ASR. Also, these PIs and differential varieties were tested against a panel of monosporic isolates. Plants presenting tan lesions were classified as susceptible (S) while plants presenting reddish-brown (RB) lesions were classified as resistant. DNA bulks were genotyped with Infinium BeadChips and the genomic region identified was further analyzed in the F2 individuals with target GBS (tGBS). PI 594,56 presented a unique resistance profile compared to the differential varieties. The resistance was monogenic dominant; however, it was classified as incompletely dominant when quantitatively studied. Genetic and QTL mapping placed the PI 594756 gene between the genomic region located at 55,863,741 and 56,123,516 bp of chromosome 18. This position is slightly upstream mapping positions of Rpp1 (PI 200492) and Rpp1-b (PI 594538A). Finally, we performed a haplotype analysis in a whole genomic sequencing-SNP database composed of Brazilian historical germplasm and sources of Rpp genes. We found SNPs that successfully differentiated the new PI 594756 allele from Rpp1 and Rpp1-b sources. The haplotype identified can be used as a tool for marker-assisted selection (MAS). Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-023-01358-4.

Overexpression of the GmEXPA1 gene reduces plant susceptibility to Meloidogyne incognita.

KEY MESSAGE: The overexpression of the soybean GmEXPA1 gene reduces plant susceptibility to M. incognita by the increase of root lignification. Plant expansins are enzymes that act in a pH-dependent manner in the plant cell wall loosening and are associated with improved tolerance or resistance to abiotic or biotic stresses. Plant-parasitic nematodes (PPN) can alter the expression profile of several expansin genes in infected root cells. Studies have shown that overexpression or downregulation of particular expansin genes can reduce plant susceptibility to PPNs. Root-knot nematodes (RKN) are obligate sedentary endoparasites of the genus Meloidogyne spp. of which M. incognita is one of the most reported species. Herein, using a transcriptome dataset and real-time PCR assays were identified an expansin A gene (GmEXPA1; Glyma.02G109100) that is upregulated in the soybean nematode-resistant genotype PI595099 compared to the susceptible cultivar BRS133 during plant parasitism by M. incognita. To understand the role of the GmEXPA1 gene during the interaction between soybean plant and M. incognita were generated stable A. thaliana and N. tabacum transgenic lines. Remarkably, both A. thaliana and N. tabacum transgenic lines overexpressing the GmEXPA1 gene showed reduced susceptibility to M. incognita. Furthermore, plant growth, biomass accumulation, and seed yield were not affected in these transgenic lines. Interestingly, significant upregulation of the NtACC oxidase and NtEFE26 genes, involved in ethylene biosynthesis, and NtCCR and Nt4CL genes, involved in lignin biosynthesis, was observed in roots of the N. tabacum transgenic lines, which also showed higher lignin content. These data suggested a possible link between GmEXPA1 gene expression and increased lignification of the root cell wall. Therefore, these data support that engineering of the GmEXPA1 gene in soybean offers a powerful biotechnology tool to assist in RKN management.

A Phakopsora pachyrhizi Effector Suppresses PAMP-Triggered Immunity and Interacts with a Soybean Glucan Endo-1,3-ß-Glucosidase to Promote Virulence.

Asian soybean rust, caused by the fungus Phakopsora pachyrhizi, is one of the most important diseases affecting soybean production in tropical areas. During infection, P. pachyrhizi secretes proteins from haustoria that are transferred into plant cells to promote virulence. To date, only one candidate P. pachyrhizi effector protein has been characterized in detail to understand the mechanism by which it suppresses plant defenses to enhance infection. Here, we aimed to extend understanding of the pathogenic mechanisms of P. pachyrhizi based on the discovery of host proteins that interact with the effector candidate Phapa-7431740. We demonstrated that Phapa-7431740 suppresses pathogen-associated molecular pattern-triggered immunity (PTI) and that it interacts with a soybean glucan endo-1,3-ß-glucosidase (GmßGLU), a pathogenesis-related (PR) protein belonging to the PR-2 family. Structural and phylogenetic characterization of the PR-2 protein family predicted in the soybean genome and comparison to PR-2 family members in Arabidopsis thaliana and cotton, demonstrated that GmßGLU is a type IV ß-1,3-glucanase. Transcriptional profiling during an infection time course showed that the GmßGLU mRNA is highly induced during the initial hours after infection, coinciding with peak of expression of Phapa-7431740. The effector was able to interfere with the activity of GmßGLU in vitro, with a dose-dependent inhibition. Our results suggest that Phapa-7431740 may suppress PTI by interfering with glucan endo-1,3-ß-glucosidase activity. [Formula: 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.

Overexpression of a soybean Globin (GmGlb1-1) gene reduces plant susceptibility to Meloidogyne incognita.

MAIN CONCLUSION: The overexpression of the GmGlb1-1 gene reduces plant susceptibility to Meloidogyne incognita. Non-symbiotic globin class #1 (Glb1) genes are expressed in different plant organs, have a high affinity for oxygen, and are related to nitric oxide (NO) turnover. Previous studies showed that soybean Glb1 genes are upregulated in soybean plants under flooding conditions. Herein, the GmGlb1-1 gene was identified in soybean as being upregulated in the nematode-resistant genotype PI595099 compared to the nematode-susceptible cultivar BRS133 during plant parasitism by Meloidogyne incognita. The Arabidopsis thaliana and Nicotiana tabacum transgenic lines overexpressing the GmGlb1-1 gene showed reduced susceptibility to M. incognita. Consistently, gall morphology data indicated that pJ2 nematodes that infected the transgenic lines showed developmental alterations and delayed parasitism progress. Although no significant changes in biomass and seed yield were detected, the transgenic lines showed an elongated, etiolation-like growth under well-irrigation, and also developed more axillary roots under flooding conditions. In addition, transgenic lines showed upregulation of some important genes involved in plant defense response to oxidative stress. In agreement, higher hydrogen peroxide accumulation and reduced activity of reactive oxygen species (ROS) detoxification enzymes were also observed in these transgenic lines. Thus, based on our data and previous studies, it was hypothesized that constitutive overexpression of the GmGlb1-1 gene can interfere in the dynamics of ROS production and NO scavenging, enhancing the acquired systemic acclimation to biotic and abiotic stresses, and improving the cellular homeostasis. Therefore, these collective data suggest that ectopic or nematode-induced overexpression, or enhanced expression of the GmGlb1-1 gene using CRISPR/dCas9 offers great potential for application in commercial soybean cultivars aiming to reduce plant susceptibility to M. incognita.

Genome-wide association study for resistance to the Meloidogyne javanica causing root-knot nematode in soybean.

KEY MESSAGE: A locus on chromosome 13, containing multiple TIR-NB-LRR genes and SNPs associated with M. javanica resistance, was identified using a combination of GWAS, resequencing, genetic mapping and expression profiling. Meloidogyne javanica, a root-knot nematode, is an important problem in soybean-growing areas, leading to severe yield losses. Some accessions have been identified carrying resistance loci to this nematode. In this study, a set of 317 soybean accessions was characterized for resistance to M. javanica. A genome-wide association study was performed using SNPs from genotyping-by-sequencing, and a region of 29.2 kb on chromosome 13 was identified. An analysis of haplotypes showed that SNPs were able to discriminate between susceptible and resistant accessions, with 25 accessions sharing the haplotype associated with resistance. Furthermore, five accessions that exhibited resistance without carrying this haplotype may carry different loci conferring resistance to M. javanica. We also conducted the screening of the SNPs in the USDA soybean germplasm, revealing that several soybean accessions previously reported as resistant to other nematodes also shared the resistance haplotype on chromosome 13. Two SNP-based TaqMan® assays were developed and validated in two panels of soybean cultivars and in biparental populations. In silico analysis of the region associated with resistance identified the occurrence of genes with structural similarity with classical major resistance genes (NBS-LRR genes). Specifically, several nonsynonymous SNPs were observed in Glyma.13g194800 and Glyma.13g194900. The expression profile of these candidate genes demonstrated that the two gene models were up-regulated in the resistance source PI 505,099 after nematode infection. Overall, the SNPs associated with resistance and the genes identified constitute an important tool for introgression of resistance to the root-knot nematode by marker-assisted selection in soybean breeding programs.

The soybean gene GmHsp22.4 is involved in the resistance response to Meloidogyne javanica in Arabidopsis thaliana.

BACKGROUND: Small heat shock proteins (sHSPs) belong to the class of molecular chaperones that respond to biotic and abiotic stresses in plants. A previous study has showed strong induction of the gene GmHsp22.4 in response to the nematode Meloidogyne javanica in a resistant soybean genotype, while repression in a susceptible one. This study aimed to investigate the functional involvement of this small chaperone in response to M. javanica in Arabidopsis thaliana. First, it was evaluated the activation of the promoter region after the nematode inoculation, and the occurrence of polymorphisms between resistant and susceptible re-sequenced soybean accessions. Then functional analysis using A. thaliana lines overexpressing the soybean GmHsp22.4 gene, and knocked-out mutants were challenged with M. javanica infestation. RESULTS: High expression levels of the GFP gene marker in transformed A. thaliana plants revealed that the promoter region of GmHsp22.4 was strongly activated after nematode inoculation. Moreover, the multiplication of the nematode was significantly reduced in plants overexpressing GmHsp22.4 gene in A. thaliana compared to the wild type. Additionally, the multiplication of M. javanica in the A. thaliana mutants was significantly increased mainly in the event athsp22.0-2. This increase was not that evident in the event athsp22.0-1, the one that preserved a portion of the promoter region, including the HSEs in the region around - 83 bp. However, structural analysis at sequence level among soybean resistant and susceptible genotypes did not detect any polymorphisms in the whole gene model. CONCLUSIONS: The soybean chaperone GmHsp22.4 is involved in the defense response to root-knot nematode M. javanica in A. thaliana. Specifically, the promoter region covering until - 191 from the transcriptional start site (TSS) is necessary to promoter activation after nematode infection in Arabidopsis. No polymorphisms that could explain these differences in the defense response were detected in the GmHsp22.4 gene between resistant and susceptible soybean genotypes. Therefore, further investigation is needed to elucidate the triggering factor of the plant's defense mechanism, both at the sequence level of the soybean genotypes presenting contrasting reaction to root-knot nematode and by detecting cis-elements that are essential for the activation of the GmHsp22.4 gene promoter.

Transcriptional profile of genes involved in the production of terpenes and glyceollins in response to biotic stresses in soybean.

Terpenes produced by plants comprise a diverse range of secondary metabolites, including volatile organic compounds (VOCs). Terpene VOC production may be altered after damage or by biological stimuli such as bacterial, fungal and insects, and subsequent triggering of plant defense responses. These VOCs originate in plants from two independent pathways: the mevalonate and the methylerythritol phosphate pathways, which utilize dimethylallyl and isopentenyl diphosphates to form the terpenoidal precursors. Phakopsora pachyrhizi fungi causes Asian soybean rust, limiting soybean production and resulting in losses of up to 80% if no control strategies are applied. By using a transcriptome datasets, we investigated the regulation of genes of the mevalonate pathway under different biotic stresses. We studied the impact of P. pachyrhizi infection in vivo expression profile of genes involved in terpenoid and glyceollin biosynthesis in genotypes harboring different resistance genes (Rpp), and across the infection cycle. In addition, we used UPLC and UPGC analysis to evaluate glyceollin and VOC production, respectively, to identify metabolites associated with soybean responses to pathogen infection. The regulation of soybean genes involved in terpene production was influenced by genotypes, depending on the Rpp gene, while glyceollin was induced in all genotypes. Furthermore, a sesquiterpene was identified as a potential marker associated with rust symptoms on soybean.

Association mapping of a locus that confers southern stem canker resistance in soybean and SNP marker development.

BACKGROUND: Southern stem canker (SSC), caused by Diaporthe aspalathi (E. Jansen, Castl. & Crous), is an important soybean disease that has been responsible for severe losses in the past. The main strategy for controlling this fungus involves the introgression of resistance genes. Thus far, five main loci have been associated with resistance to SSC. However, there is a lack of information about useful allelic variation at these loci. In this work, a genome-wide association study (GWAS) was performed to identify allelic variation associated with resistance against Diaporthe aspalathi and to provide molecular markers that will be useful in breeding programs. RESULTS: We characterized the response to SSC infection in a panel of 295 accessions from different regions of the world, including important Brazilian elite cultivars. Using a GBS approach, the panel was genotyped, and we identified marker loci associated with Diaporthe aspalathi resistance through GWAS. We identified 19 SNPs associated with southern stem canker resistance, all on chromosome 14. The peak SNP showed an extremely high degree of association (p-value = 6.35E-27) and explained a large amount of the observed phenotypic variance (R2 = 70%). This strongly suggests that a single major gene is responsible for resistance to D. aspalathi in most of the lines constituting this panel. In resequenced soybean materials, we identified other SNPs in the region identified through GWAS in the same LD block that clearly differentiate resistant and susceptible accessions. The peak SNP was selected and used to develop a cost-effective molecular marker assay, which was validated in a subset of the initial panel. In an accuracy test, this SNP assay demonstrated 98% selection efficiency. CONCLUSIONS: Our results suggest relevance of this locus to SSC resistance in soybean cultivars and accessions from different countries, and the SNP marker assay developed in this study can be directly applied in MAS studies in breeding programs to select materials that are resistant against this pathogen and support its introgression.

New insights into Phakopsora pachyrhizi infection based on transcriptome analysis in planta.

Asian soybean rust (ASR) is one of the most destructive diseases affecting soybeans. The causative agent of ASR, the fungus Phakopsora pachyrhizi, presents characteristics that make it difficult to study in vitro, limiting our knowledge of plant-pathogen dynamics. Therefore, this work used leaf lesion laser microdissection associated with deep sequencing to determine the pathogen transcriptome during compatible and incompatible interactions with soybean. The 36,350 generated unisequences provided an overview of the main genes and biological pathways that were active in the fungus during the infection cycle. We also identified the most expressed transcripts, including sequences similar to other fungal virulence and signaling proteins. Enriched P. pachyrhizi transcripts in the resistant (PI561356) soybean genotype were related to extracellular matrix organization and metabolic signaling pathways and, among infection structures, in amino acid metabolism and intracellular transport. Unisequences were further grouped into gene families along predicted sequences from 15 other fungi and oomycetes, including rust fungi, allowing the identification of conserved multigenic families, as well as being specific to P. pachyrhizi. The results revealed important biological processes observed in P. pachyrhizi, contributing with information related to fungal biology and, consequently, a better understanding of ASR.

Evaluation of genetic variation among Brazilian soybean cultivars through genome resequencing.

BACKGROUND: Soybean [Glycine max (L.) Merrill] is one of the most important legumes cultivated worldwide, and Brazil is one of the main producers of this crop. Since the sequencing of its reference genome, interest in structural and allelic variations of cultivated and wild soybean germplasm has grown. To investigate the genetics of the Brazilian soybean germplasm, we selected soybean cultivars based on the year of commercialization, geographical region and maturity group and resequenced their genomes. RESULTS: We resequenced the genomes of 28 Brazilian soybean cultivars with an average genome coverage of 14.8X. A total of 5,835,185 single nucleotide polymorphisms (SNPs) and 1,329,844 InDels were identified across the 20 soybean chromosomes, with 541,762 SNPs, 98,922 InDels and 1,093 CNVs that were exclusive to the 28 Brazilian cultivars. In addition, 668 allelic variations of 327 genes were shared among all of the Brazilian cultivars, including genes related to DNA-dependent transcription-elongation, photosynthesis, ATP synthesis-coupled electron transport, cellular respiration, and precursors of metabolite generation and energy. A very homogeneous structure was also observed for the Brazilian soybean germplasm, and we observed 41 regions putatively influenced by positive selection. Finally, we detected 3,880 regions with copy-number variations (CNVs) that could help to explain the divergence among the accessions evaluated. CONCLUSIONS: The large number of allelic and structural variations identified in this study can be used in marker-assisted selection programs to detect unique SNPs for cultivar fingerprinting. The results presented here suggest that despite the diversification of modern Brazilian cultivars, the soybean germplasm remains very narrow because of the large number of genome regions that exhibit low diversity. These results emphasize the need to introduce new alleles to increase the genetic diversity of the Brazilian germplasm.

Differential expression of four soybean bZIP genes during Phakopsora pachyrhizi infection.

Asian soybean rust (ASR), caused by the obligate biotrophic fungus Phakopsora pachyrhizi, is one of most important diseases in the soybean (Glycine max (L.) Merr.) agribusiness. The identification and characterization of genes related to plant defense responses to fungal infection are essential to develop ASR-resistant plants. In this work, we describe four soybean genes, GmbZIP62, GmbZIP105, GmbZIPE1, and GmbZIPE2, which encode transcription factors containing a basic leucine zipper (bZIP) domain from two divergent classes, and that are responsive to P. pachyrhizi infection. Molecular phylogenetic analyses demonstrated that these genes encode proteins similar to bZIP factors responsive to pathogens. Yeast transactivation assays showed that only GmbZIP62 has strong transactivation activity in yeast. In addition, three of the bZIP transcription factors analyzed were also differentially expressed by plant defense hormones, and all were differentially expressed by fungal attack, indicating that these proteins might participate in response to ASR infection. The results suggested that these bZIP proteins are part of the plant defense response to P. pachyrhizi infection, by regulating the gene expression related to ASR infection responses. These bZIP genes are potential targets to obtain new soybean genotypes resistant to ASR.

Positive and negative roles for soybean MPK6 in regulating defense responses.

It has been well established that MPK6 is a positive regulator of defense responses in model plants such as Arabidopsis and tobacco. However, the functional importance of soybean MPK6 in disease resistance has not been investigated. Here, we showed that silencing of GmMPK6 in soybean using virus-induced gene silencing mediated by Bean pod mottle virus (BPMV) caused stunted growth and spontaneous cell death on the leaves, a typical phenotype of activated defense responses. Consistent with this phenotype, expression of pathogenesis-related (PR) genes and the conjugated form of salicylic acid were significantly increased in GmMPK6-silenced plants. As expected, GmMPK6-silenced plants were more resistant to downy mildew and Soybean mosaic virus compared with vector control plants, indicating a negative role of GmMPK6 in disease resistance. Interestingly, overexpression of GmMPK6, either transiently in Nicotiana benthamiana or stably in Arabidopsis, resulted in hypersensitive response (HR)-like cell death. The HR-like cell death was accompanied by increased PR gene expression, suggesting that GmMPK6, like its counterpart in other plant species, also plays a positive role in cell death induction and defense response. Using bimolecular fluorescence complementation analysis, we determined that GmMKK4 might function upstream of GmMPK6 and GmMKK4 could interact with GmMPK6 independent of its phosphorylation status. Taken together, our results indicate that GmMPK6 functions as both repressor and activator in defense responses of soybean.

Genome-wide annotation of the soybean WRKY family and functional characterization of genes involved in response to Phakopsora pachyrhizi infection.

BACKGROUND: Many previous studies have shown that soybean WRKY transcription factors are involved in the plant response to biotic and abiotic stresses. Phakopsora pachyrhizi is the causal agent of Asian Soybean Rust, one of the most important soybean diseases. There are evidences that WRKYs are involved in the resistance of some soybean genotypes against that fungus. The number of WRKY genes already annotated in soybean genome was underrepresented. In the present study, a genome-wide annotation of the soybean WRKY family was carried out and members involved in the response to P. pachyrhizi were identified. RESULTS: As a result of a soybean genomic databases search, 182 WRKY-encoding genes were annotated and 33 putative pseudogenes identified. Genes involved in the response to P. pachyrhizi infection were identified using superSAGE, RNA-Seq of microdissected lesions and microarray experiments. Seventy-five genes were differentially expressed during fungal infection. The expression of eight WRKY genes was validated by RT-qPCR. The expression of these genes in a resistant genotype was earlier and/or stronger compared with a susceptible genotype in response to P. pachyrhizi infection. Soybean somatic embryos were transformed in order to overexpress or silence WRKY genes. Embryos overexpressing a WRKY gene were obtained, but they were unable to convert into plants. When infected with P. pachyrhizi, the leaves of the silenced transgenic line showed a higher number of lesions than the wild-type plants. CONCLUSIONS: The present study reports a genome-wide annotation of soybean WRKY family. The participation of some members in response to P. pachyrhizi infection was demonstrated. The results contribute to the elucidation of gene function and suggest the manipulation of WRKYs as a strategy to increase fungal resistance in soybean plants.

Potential fate of ingested Lactobacillus plantarum and its occurrence in human feces.

Lactobacillus plantarum has been used in human clinical trials to promote beneficial effects in the immune system, to alleviate intestinal disorders, and to reduce the risk of cardiovascular disease. It is also involved in many fermentation processes in the food industry. However, information on the fate of ingested L. plantarum is limited. In this study, 61 subjects received daily doses of fermented milk containing 2 × 10(11) cells of L. plantarum Lp115 for different periods of time. The target microorganism was monitored in the fecal microbiota via quantitative PCR (qPCR). L. plantarum was detected and quantified in all of the subjects during the ingestion periods. The differences between the L. plantarum levels at time zero and during all the different ingestion periods were statistically significant (P = 0.001). However, at 15 and 45 days after discontinuing supplementation, the number of lactobacilli was reduced to the baseline level (those at time zero). A longer period with L. plantarum in the diet did not result in increased levels of this bacterium in the stool, based on postconsumption evaluations (P = 0.001). The qPCR method was specific and sensitive for L. plantarum quantification in such a complex microbial environment as the gastrointestinal tract.

Phenotyping soybean plants transformed with rd29A:AtDREB1A for drought tolerance in the greenhouse and field.

The development of drought tolerant plants is a high priority because the area suffering from drought is expected to increase in the future due to global warming. One strategy for the development of drought tolerance is to genetically engineer plants with transcription factors (TFs) that regulate the expression of several genes related to abiotic stress defense responses. This work assessed the performance of soybean plants overexpressing the TF DREB1A under drought conditions in the field and in the greenhouse. Drought was simulated in the greenhouse by progressively drying the soil of pot cultures of the P58 and P1142 lines. In the field, the performance of the P58 line and of 09D-0077, a cross between the cultivars BR16 and P58, was evaluated under four different water regimes: irrigation, natural drought (no irrigation) and water stress created using rain-out shelters in the vegetative or reproductive stages. Although the dehydration-responsive element-binding protein (DREB) plants did not outperform the cultivar BR16 in terms of yield, some yield components were increased when drought was introduced during the vegetative stage, such as the number of seeds, the number of pods with seeds and the total number of pods. The greenhouse data suggest that the higher survival rates of DREB plants are because of lower water use due to lower transpiration rates under well watered conditions. Further studies are needed to better characterize the soil and atmospheric conditions under which these plants may outperform the non-transformed parental plants.

Transcriptional analysis of genes involved in nodulation in soybean roots inoculated with Bradyrhizobium japonicum strain CPAC 15.

BACKGROUND: Biological nitrogen fixation in root nodules is a process of great importance to crops of soybean [Glycine max (L.) Merr.], as it may provide the bulk of the plant's needs for nitrogen. Legume nodulation involves several complex steps and, although studied for many decades, much remains to be understood. RESULTS: This research aimed at analyzing the global expression of genes in soybean roots of a Brazilian cultivar (Conquista) inoculated with Bradyrhizobium japonicum CPAC 15, a strain broadly used in commercial inoculants in Brazil. To achieve this, we used the suppressive subtractive hybridization (SSH) technique combined with Illumina sequencing. The subtractive library (non-inoculated x inoculated) of soybean roots resulted in 3,210 differentially expressed transcripts at 10 days after inoculation were studied. The data were grouped according to the ontologies of the molecular functions and biological processes. Several classes of genes were confirmed as related to N2 fixation and others were reported for the first time. CONCLUSIONS: During nodule formation, a higher percentage of genes were related to primary metabolism, cell-wall modifications and the antioxidant defense system. Putative symbiotic functions were attributed to some of these genes for the first time.

Genome-wide analysis of the Hsp20 gene family in soybean: comprehensive sequence, genomic organization and expression profile analysis under abiotic and biotic stresses.

BACKGROUND: The Hsp20 genes are associated with stress caused by HS and other abiotic factors, but have recently been found to be associated with the response to biotic stresses. These genes represent the most abundant class among the HSPs in plants, but little is known about this gene family in soybean. Because of their apparent multifunctionality, these proteins are promising targets for developing crop varieties that are better adapted to biotic and abiotic stresses. Thus, in the present study an in silico identification of GmHsp20 gene family members was performed, and the genes were characterized and subjected to in vivo expression analysis under biotic and abiotic stresses. RESULTS: A search of the available soybean genome databases revealed 51 gene models as potential GmHsp20 candidates. The 51 GmHsp20 genes were distributed across a total of 15 subfamilies where a specific predicted secondary structure was identified. Based on in vivo analysis, only 47 soybean Hsp20 genes were responsive to heat shock stress. Among the GmHsp20 genes that were potentials HSR, five were also cold-induced, and another five, in addition to one GmAcd gene, were responsive to Meloidogyne javanica infection. Furthermore, one predicted GmHsp20 was shown to be responsive only to nematode infection; no expression change was detected under other stress conditions. Some of the biotic stress-responsive GmHsp20 genes exhibited a divergent expression pattern between resistant and susceptible soybean genotypes under M. javanica infection. The putative regulatory elements presenting some conservation level in the GmHsp20 promoters included HSE, W-box, CAAT box, and TA-rich elements. Some of these putative elements showed a unique occurrence pattern among genes responsive to nematode infection. CONCLUSIONS: The evolution of Hsp20 family in soybean genome has most likely involved a total of 23 gene duplications. The obtained expression profiles revealed that the majority of the 51 GmHsp20 candidates are induced under HT, but other members of this family could also be involved in normal cellular functions, unrelated to HT. Some of the GmHsp20 genes might be specialized to respond to nematode stress, and the predicted promoter structure of these genes seems to have a particular conserved pattern related to their biological function.

Fast induction of biosynthetic polysaccharide genes lpxA, lpxE, and rkpI of Rhizobium sp. strain PRF 81 by common bean seed exudates is indicative of a key role in symbiosis.

Rhizobial surface polysaccharides (SPS) are, together with nodulation (Nod) factors, recognized as key molecules for establishment of rhizobia-legume symbiosis. In Rhizobium tropici, an important nitrogen-fixing symbiont of common bean (Phaseolus vulgaris L.), molecular structures and symbiotic roles of the SPS are poorly understood. In this study, Rhizobium sp. strain PRF 81 genes, belonging to the R. tropici group, were investigated: lpxA and lpxE, involved in biosynthesis and modification of the lipid-A anchor of lipopolysaccharide (LPS), and rkpI, involved in synthesis of a lipid carrier required for production of capsular polysaccharides (KPS). Reverse transcription quantitative PCR (RT-qPCR) analysis revealed, for the first time, that inducers released from common bean seeds strongly stimulated expression of all three SPS genes. When PRF 81 cells were grown for 48 h in the presence of seed exudates, twofold increases (p < 0.05) in the transcription levels of lpxE, lpxA, and rkpI genes were observed. However, higher increases (p < 0.05) in transcription rates, about 50-fold for lpxE and about 30-fold for lpxA and rkpI, were observed after only 5 min of incubation with common bean seed exudates. Evolutionary analyses revealed that lpxA and lpxE of PRF81 and of the type strain of R. tropici CIAT899(T)clustered with orthologous Rhizobium radiobacter and were more related to R. etli and Rhizobium leguminosarum, while rkpI was closer to the Sinorhizobium sp. group. Upregulation of lpxE, lpxA, and rkpI genes suggests that seed exudates can modulate production of SPS of Rhizobium sp. PRF81, leading to cell wall changes necessary for symbiosis establishment.

The Lesion Simulating Disease (LSD) gene family as a variable in soybean response to Phakopsora pachyrhizi infection and dehydration.

The Lesion Simulating Disease (LSD) genes encode a family of zinc finger proteins that are reported to play an important role in the hypersensitive response and programmed cell death (PCD) that are caused by biotic and abiotic stresses. In the present study, 117 putative LSD family members were identified in Viridiplantae. Genes with one, two, or three conserved LSD domains were identified. Proteins with three LSD domains were highly represented in the species analyzed and were present in basal organisms. Proteins with two LSD domains were identified only in the Embryophyte clade, and proteins possessing one LSD domain were highly represented in grass species. Expression analyses of Glycine max LSD (GmLSD) genes were performed by real-time quantitative polymerase chain reaction. The results indicated that GmLSD genes are not ubiquitously expressed in soybean organs and that their expression patterns are instead organ-dependent. The expression of the majority of GmLSD genes is modulated in soybean during Phakopsora pachyrhizi infection. In addition, the expression of some GmLSD genes is modulated in plants under dehydration stress. These results suggest the involvement of GmLSD genes in the response of soybean to both biotic and abiotic stresses.