Roles of small RNAs in soybean defense against Phytophthora sojae infection

The genus Phytophthora consists of many notorious pathogens of crops and forestry trees. At present, battling Phytophthora diseases is challenging due to a lack of understanding of their pathogenesis. We investigated the role of small RNAs in regulating soybean defense in response to infection by Phytophthora sojae, the second most destructive pathogen of soybean. Small RNAs, including microRNAs (miRNAs) and small interfering RNAs (siRNAs), are universal regulators that repress target gene expression in eukaryotes. We identified known and novel small RNAs that differentially accumulated during P. sojae infection in soybean roots. Among them, miR393 and miR166 were induced by heat‐inactivated P. sojae hyphae, indicating that they may be involved in soybean basal defense. Indeed, knocking down the level of mature miR393 led to enhanced susceptibility of soybean to P. sojae; furthermore, the expression of isoflavonoid biosynthetic genes was drastically reduced in miR393 knockdown roots. These data suggest that miR393 promotes soybean defense against P. sojae. In addition to miRNAs, P. sojae infection also resulted in increased accumulation of phased siRNAs (phasiRNAs) that are predominantly generated from canonical resistance genes encoding nucleotide binding‐leucine rich repeat proteins and genes encoding pentatricopeptide repeat‐containing proteins. This work identifies specific miRNAs and phasiRNAs that regulate defense‐associated genes in soybean during Phytophthora infection.     

Genome-Wide Association Study of Partial Resistance to P. sojae in Wild Soybeans from Heilongjiang Province,China

Phytophthora root rot (PRR) is a destructive disease of soybeans (Glycine max (L.) Merr) caused by Phytophthora sojae (P. sojae). The most effective way to prevent the disease is growing resistant or tolerant varieties. Partial resistance provides a more durable resistance against the pathogen compared to complete resistance. Wild soybean (Glycine soja Sieb. & Zucc.) seems to be an extraordinarily important gene pool for soybean improvement due to its high level of genetic variation. In this study, 242 wild soybean germplasms originating from different regions of Heilongjiang province were used to identify resistance genes to P. sojae race 1 using a genome-wide association study (GWAS). A total of nine significant SNPs were detected, repeatedly associated with P. sojae resistance and located on chromosomes 1, 10, 12, 15, 17, 19 and 20. Among them, seven favorable allelic variations associated with P. sojae resistance were evaluated by a t-test. Eight candidate genes were predicted to explore the mechanistic hypotheses of partial resistance, including Glysoja.19G051583, which encodes an LRR receptor-like serine/threonine protein kinase protein, Glysoja.19G051581, which encodes a receptor-like cytosolic serine/threonine protein kinase protein. These findings will provide additional insights into the genetic architecture of P. sojae resistance in a large sample of wild soybeans and P. sojae-resistant breeding through marker-assisted selection.     

A Phytophthora sojae Glycoside Hydrolase 12 Protein Is a Major Virulence Factor during Soybean Infection and Is Recognized as a PAMP

We identified a glycoside hydrolase family 12 (GH12) protein, XEG1, produced by the soybean pathogen Phytophthora sojae that exhibits xyloglucanase and β-glucanase activity. It acts as an important virulence factor during P. sojae infection but also acts as a pathogen-associated molecular pattern (PAMP) in soybean (Glycine max) and solanaceous species, where it can trigger defense responses including cell death. GH12 proteins occur widely across microbial taxa, and many of these GH12 proteins induce cell death in Nicotiana benthamiana. The PAMP activity of XEG1 is independent of its xyloglucanase activity. XEG1 can induce plant defense responses in a BAK1-dependent manner. The perception of XEG1 occurs independently of the perception of ethylene-inducing xylanase. XEG1 is strongly induced in P. sojae within 30 min of infection of soybean and then slowly declines. Both silencing and overexpression of XEG1 in P. sojae severely reduced virulence. Many P. sojae RXLR effectors could suppress defense responses induced by XEG1, including several that are expressed within 30 min of infection. Therefore, our data suggest that PsXEG1 contributes to P. sojae virulence, but soybean recognizes PsXEG1 to induce immune responses, which in turn can be suppressed by RXLR effectors. XEG1 thus represents an apoplastic effector that is recognized via the plant’s PAMP recognition machinery.     

Isolate Identity Determines Plant Tolerance to Pathogen Attack in Assembled Mycorrhizal Communities

Arbuscular mycorrhizal fungi (AMF) are widespread soil microorganisms that associate mutualistically with plant hosts. AMF receive photosynthates from the host in return for various benefits. One of such benefits is in the form of enhanced pathogen tolerance. However, this aspect of the symbiosis has been understudied compared to effects on plant growth and its ability to acquire nutrients. While it is known that increased AMF species richness positively correlates with plant productivity, the relationship between AMF diversity and host responses to pathogen attack remains obscure. The objective of this study was to test whether AMF isolates can differentially attenuate the deleterious effects of a root pathogen on plant growth, whether the richest assemblage of AMF isolates provides the most tolerance against the pathogen, and whether AMF-induced changes to root architecture serve as a mechanism for improved plant disease tolerance. In a growth chamber study, we exposed the plant oxeye daisy (Leucanthemum vulgare) to all combinations of three AMF isolates and to the plant root pathogen Rhizoctonia solani. We found that the pathogen caused an 81% reduction in shoot and a 70% reduction in root biomass. AMF significantly reduced the highly deleterious effect of the pathogen. Mycorrhizal plants infected with the pathogen produced 91% more dry shoot biomass and 72% more dry root biomass relative to plants solely infected with R. solani. AMF isolate identity was a better predictor of AMF-mediated host tolerance to the pathogen than AMF richness. However, the enhanced tolerance response did not result from AMF-mediated changes to root architecture. Our data indicate that AMF communities can play a major role in alleviating host pathogen attack but this depends primarily on the capacity of individual AMF isolates to provide this benefit.     

Arabidopsis non-host resistance PSS30 gene enhances broad-spectrum disease resistance in the soybean cultivar Williams 82

Non-host resistance (NHR), which protects all members of a plant species from non-adapted or non-host plant pathogens, is the most common form of plant immunity. NHR provides the most durable and robust form of broad-spectrum immunity against non-adaptive pathogens pathogenic to other crop species. In a mutant screen for loss of Arabidopsis (Arabidopsis thaliana) NHR against the soybean (Glycine max (L.) Merr.) pathogen Phytophthora sojae, the Phytophthora sojae-susceptible 30 (pss30) mutant was identified. The pss30 mutant is also susceptible to the soybean pathogen Fusarium virguliforme. PSS30 encodes a folate transporter, AtFOLT1, which was previously localized to chloroplasts and implicated in the transport of folate from the cytosol to plastids. We show that two Arabidopsis folate biosynthesis mutants with reduced folate levels exhibit a loss of non-host immunity against P. sojae. As compared to the wild-type Col-0 ecotype, the steady-state folate levels are reduced in the pss1, atfolt1 and two folate biosynthesis mutants, suggesting that folate is required for non-host immunity. Overexpression of AtFOLT1 enhances immunity of transgenic soybean lines against two serious soybean pathogens, the fungal pathogen F. virguliforme and the soybean cyst nematode (SCN) Heterodera glycines. Transgenic lines showing enhanced SCN resistance also showed increased levels of folate accumulation. This study thus suggests that folate contributes to non-host plant immunity and that overexpression of a non-host resistance gene could be a suitable strategy for generating broad-spectrum disease resistance in crop plants.     

Isolation of ethylene-insensitive soybean mutants that are altered in pathogen susceptibility and gene-for-gene disease resistance

Plants commonly respond to pathogen infection by increasing ethylene production, but it is not clear if this ethylene does more to promote disease susceptibility or disease resistance. Ethylene production and/or responsiveness can be altered by genetic manipulation. The present study used mutagenesis to identify soybean (Glycine max L. Merr.) lines with reduced sensitivity to ethylene. Two new genetic loci were identified, Etr1 and Etr2. Mutants were compared with isogenic wild-type parents for their response to different soybean pathogens. Plant lines with reduced ethylene sensitivity developed similar or less-severe disease symptoms in response to virulent Pseudomonas syringae pv glycinea and Phytophthora sojae, but some of the mutants developed similar or more-severe symptoms in response to Septoria glycines and Rhizoctonia solani. Gene-for-gene resistance against P. syringae expressing avrRpt2 remained effective, but Rps1-k-mediated resistance against P. sojae races 4 and 7 was disrupted in the strong ethylene-insensitive etr1-1 mutant. Rps1-k-mediated resistance against P. sojae race 1 remained effective, suggesting that the Rps1-k locus may encode more than one gene for disease resistance. Overall, our results suggest that reduced ethylene sensitivity can be beneficial against some pathogens but deleterious to resistance against other pathogens.     

Aquaporin genes GintAQPF1 and GintAQPF2 from Glomus intraradices contribute to plant drought tolerance

Arbuscular mycorrhizal (AM) symbiosis, established between AM fungi (AMF) and roots of higher plants, occurs in most terrestrial ecosystems. It has been well demonstrated that AM symbiosis can improve plant performance under various environmental stresses, including drought stress. However, the molecular basis for the direct involvement of AMF in plant drought tolerance has not yet been established. Most recently, we cloned two functional aquaporin genes, GintAQPF1 and GintAQPF2, from AM fungus Glomus intraradices. By heterologous gene expression in yeast, aquaporin localization, activities and water permeability were examined. Gene expressions during symbiosis in expose to drought stress were also analyzed. Our data strongly supported potential water transport via AMF to host plants. As a complement, here we adopted the monoxenic culture system for AMF, in which carrot roots transformed by Ri-T DNA were cultured with Glomus intraradices in two-compartment Petri dishes, to verify the aquaporin gene functions in assisting AMF survival under polyethylene glycol (PEG) treatment. Our results showed that 25% PEG significantly upregulated the expression of two aquaporin genes, which was in line with the gene functions examined in yeast. We therefore concluded that the aquaporins function similarly in AMF as in yeast subjected to osmotic stress. The study provided further evidence to the direct involvement of AMF in improving plant water relations under drought stresses.     

Over-expression of the <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> harpin-encoding gene <Emphasis Type="Italic">hrpZm</Emphasis> confers enhanced tolerance to Phytophthora root and stem rot in transgenic soybean

Phytophthora root and stem rot (PRR) caused by Phytophthora sojae is one of the most devastating diseases reducing soybean (Glycine max) production all over the world. Harpin proteins in many plant pathogenic bacteria were confirmed to enhance disease and insect resistance in crop plants. Here, a harpin protein-encoding gene hrpZpsta from the P. syringae pv. tabaci strain Psta218 was codon-optimized (renamed hrpZm) and introduced into soybean cultivars Williams 82 and Shennong 9 by Agrobacterium-mediated transformation. Three independent transgenic lines over-expressing hrpZm were obtained and exhibited stable and enhanced tolerance to P. sojae infection in T₂–T₄ generations compared to the non-transformed (NT) and empty vector (EV)-transformed plants. Quantitative real-time PCR (qRT-PCR) analysis revealed that the expression of salicylic acid-dependent genes PR1, PR12, and PAL, jasmonic acid-dependent gene PPO, and hypersensitive response (HR)-related genes GmNPR1 and RAR was significantly up-regulated after P. sojae inoculation. Moreover, the activities of defense-related enzymes such as phenylalanine ammonia lyase (PAL), polyphenoloxidase (PPO), peroxidase, and superoxide dismutase also increased significantly in the transgenic lines compared to the NT and EV-transformed plants after inoculation. Our results suggest that over-expression of the hrpZm gene significantly enhances PRR tolerance in soybean by eliciting resistance responses mediated by multiple defense signaling pathways, thus providing an alternative approach for development of soybean varieties with improved tolerance against the soil-borne pathogen PRR.     

Effects of arbuscular mycorrhiza (AM) on health ofLinum usitatissimum L. infected by fungal pathogens

Effects of arbuscular mycorrhizal (AM) symbiosis on health ofLinum usitatissimum infected by fungal pathogens were investigated exemplarily. Physiological and biochemical analyses were done to explain the mechanisms underlying the AM effects. AM plants showed increased resistance against the wilt pathogen (Fusarium oxysporum f. sp.lini), the level of this effects depended on the plant cultivars which all showed the same level of root colonization by arbuscular mycorrhizal fungi (AMF). In contrary to that, AM plants were highly susceptible against the shoot pathogenOidium lini, but they suffered less than non-AM plants in terms of shoot fresh weight, CO₂ assimilation and content of sucrose in shoot apex. This indicates that AM not only activates resistance mechanisms but also can induce tolerance against pathogens. The concentration of phytohormones such as auxin- and gibberellin-like substances were increased in shoots of AM plants. In roots the ethylene production was increased, too. Furthermore the content and composition of free sterols were highly altered in leaves of AM plants. Root infection by AMF caused an increased respiratory activity and a reduced degree of DNA methylation, but both modifications only occurred in infected root parts indicating an increasing gene activity. The presented results suggest that nearly all parts of a plant are influenced by AM but not in the same manner. In the case of mildewed linseed the effect of AM on plant health was impressing, it indicates that AM has an ability to induce tolerance.     

丛枝菌根共生体中碳、氮代谢及其相互关系

丛枝菌根共生体(arbuscular mycorrhiza, AM)是丛枝菌根真菌(arbuscular mycorrhizal fungi, AMF)与宿主植物之间形成的互惠共生形式.共生体中的碳、氮交换和代谢影响着宿主植物和共生真菌之间的营养平衡和资源重新分配,在物质和能量循环中发挥着重要作用.宿主植物光合固定的碳输送到真菌内,并且分解和释放真菌所需的生命物质和能量,包括促进孢子萌发、菌丝生长和提高氮等营养元素的吸收;而菌根真菌利用宿主植物提供的碳骨架和能量,发生氮的转化和运输,最终传递给宿主植物供其利用.本文综述了丛枝菌根共生体中碳、氮传输和代谢的主要模式,碳、氮的交互影响和调控机制,以促进丛枝菌根在可持续农业和生态系统中的应用.     

水分胁迫和杀真菌剂对黄顶菊生长和抗旱性的影响

利用盆栽试验研究水分胁迫下AM真菌对黄顶菊生长和抗旱性的影响,揭示黄顶菊入侵过程中的微生物学机制。以苯菌灵为杀真菌剂,在土壤相对含水量为120%、80%、40%和20%条件下,分别设灭菌和不灭菌两种处理。结果表明,水分胁迫显著降低了黄顶菊株高、干重和主根长,而对AM真菌侵染率无显著影响。施用苯菌灵显著降低了菌根侵染率、叶片保水力、保护酶活性、可溶性糖和可溶性蛋白含量,提高了MDA含量。不灭菌处理下黄顶菊植株对土壤有效N和有效P的利用率较高,且植株全N、P含量显著高于灭菌处理,菌根贡献率随土壤相对含水量降低而逐渐提高,重度胁迫分别是渍水条件下的1.84和1.88倍。土壤水分状况和AM真菌的交互作用对黄顶菊生物量和生理指标影响显著。AM真菌共生能够促进黄顶菊根系对土壤水分和矿质营养吸收,改善植物代谢活动,提高抗旱性。实验结果为黄顶菊合理防控措施的制定提供了依据,同时作为丛枝菌根的基础性研究也具有重要的意义。     

The Pectin Methylesterase Gene Complement of Phytophthora sojae: Structural and Functional Analyses,and the Evolutionary Relationships with Its Oomycete Homologs

Phytophthora sojae is an oomycete pathogen that causes the disease known as root and stem rot in soybean plants, frequently leading to massive economic damage. Additionally, P. sojae is increasingly being utilized as a model for phytopathogenic oomycete research. Despite the economic and scientific importance of P. sojae, the mechanism by which it penetrates the host roots is not yet fully understood. It has been found that oomycetes are not capable of penetrating the cell wall solely through mechanical force, suggesting that alternative factors facilitate breakdown of the host cell wall. Pectin methylesterases have been suggested to be important for Phytophthora pathogenicity, but no data exist on their role in the P. sojae infection process. We have scanned the newly revised version of the annotated P. sojae genome for the presence of putative pectin methylesterases genes and conducted a sequence analysis of all gene models found. We also searched for potential regulatory motifs in the promoter region of the proposed P. sojae models, and investigated the gene expression levels throughout the early course of infection on soybean plants. We found that P. sojae contains a large repertoire of pectin methylesterase-coding genes and that most of these genes display similar motifs in the promoter region, indicating the possibility of a shared regulatory mechanism. Phylogenetic analyses confirmed the evolutionary relatedness of the pectin methylesterase-coding genes within and across Phytophthora spp. In addition, the gene duplication events that led to the emergence of this gene family appear to have occurred prior to many speciation events in the genus Phytophthora. Our results also indicate that the highest levels of expression occurred in the first 24 hours post inoculation, with expression falling after this time. Our study provides evidence that pectin methylesterases may be important for the early action of the P. sojae infection process.     

Plant integrity: An important factor in plant-pathogen interactions

The effect of plant integrity and of aboveground-belowground defense signaling on plant resistance against pathogens and herbivores is emerging as a subject of scientific research. There is increasing evidence that plant defense responses to pathogen infection differ between whole intact plants and detached leaves. Studies have revealed the importance of aboveground-belowground defense signaling for plant defenses against herbivores, while our studies have uncovered that the roots as well as the plant integrity are important for the resistance of the potato cultivar Sarpo Mira against the hemibiotrophic oomycete pathogen Phytophthora infestans. Furthermore, in the Sarpo Mira–P. infestans interactions, the plant’s meristems, the stalks or both, seem to be associated with the development of the hypersensitive response and both the plant’s roots and shoots contain antimicrobial compounds when the aerial parts of the plants are infected. Here, we present a short overview of the evidence indicating the importance of plant integrity on plant defense responses.