Avirulent Pathogens Research Articles

Virulent strains of Zymoseptoria tritici suppress the host immune response and facilitate the success of avirulent strains in mixed infections

Plants interact with a plethora of pathogenic microorganisms in nature. Pathogen-plant interaction experiments focus mainly on single-strain infections, typically ignoring the complexity of multi-strain infections even though mixed infections are common and critical for the infection outcome. The wheat pathogen Zymoseptoria tritici forms highly diverse fungal populations in which several pathogen strains often colonize the same leaf. Despite the importance of mixed infections, the mechanisms governing interactions between a mixture of pathogen strains within a plant host remain largely unexplored. Here we demonstrate that avirulent pathogen strains benefit from being in mixed infections with virulent strains. We show that virulent strains suppress the wheat immune response, allowing avirulent strains to colonize the apoplast and to reproduce. Our experiments indicate that virulent strains in mixed infections can suppress the plant immune system, probably facilitating the persistence of avirulent pathogen strains in fields planted with resistant host plants.

The correlation between proline/P5C cycle and the response to avirulent pathogen infection in Arabidopsis

Proline/P5C cycle between mitochondria and cytosol, play important roles in energy supply and ROS (Reactive oxygen species) generation in mammalian cell. Recently, in plant, proline-dependent ROS via proline/P5C cycle was proposed to be involved in hypersensitive reaction during plant response to avirulent pathogens. However, much remains to be elucidated about the regulation of proline/P5C cycle upon pathogen infection. Here, we reported the isolation and characterization of an Arabidopsis mutant proline resistance 2 (pre2), with a single dominant mutation in a single gene. Our results showed that the proline resistance phenotype of pre2 is not due to decreased intracellular proline content, when treated with exogenous proline. Upon proline treatment, pre2 showed reduced induction of PDH transcript level and enhanced induction of that of P5CDH, accompanied by lower level of mitochondrial ROS, suggesting an attenuated proline/P5C cycle activity. Proline-induced SA (Salicylic acid) signaling was also less activated in pre2, as evidenced by reduced free SA content and PR1 transcript level, compared to the WT. On the other hand, SA activation on the proline/P5C activity is to a lesser extent in pre2 than in WT. Significantly, pre2 demonstrated increased susceptibility to infection by avirulent pathogen Pst. DC3000 (avrRps4), accompanied also by lesser induced proline/P5C cycle activity by the pathogen. Our results indicated that there is a correlation between proline/P5C cycle and plant response to avirulent pathogen.

RSI1/FLD and its epigenetic target RRTF1 are essential for the retention of infection memory in Arabidopsis thaliana.

Plants can retain a memory of previous pathogen infections to mount a more robust defense response during subsequent infections by developing systemic acquired resistance (SAR). However, the mechanism through which plants develop and retain infection memory is not known. Experiments have shown the association of epigenetic modifications of specific defense-related genes with SAR. RSI1/FLD codes for a histone demethylase and is required for the activation of SAR in Arabidopsis. Here we report the identification of RRTF1 as an epigenetic target of RSI1. RRTF1 expression is higher in pathogen-free distal tissues of the rsi1 mutant. Experiments with loss-of-function and overexpression lines suggest RRTF1 is a negative regulator of basal defense against virulent and avirulent pathogens as well as SAR. Enhanced expression of RRTF1 in a wild-type (WT) background specifically impairs SAR without impacting local resistance. RSI1 is recruited at the RRTF1 locus in a SAR-inducible manner and contributes to H3K4me2 and H3K4me3 demethylation. Introduction of the rrtf1 mutation rescues the loss-of-SAR phenotype of rsi1 plants. However, these plants fail to retain infection memory beyond 7 days post-primary inoculation, whereas WT plants retain memory for at least 11 days. Our results demonstrate that RSI1 and RRTF1 form a functional module for retaining infection memory in Arabidopsis.

Female scent accelerates growth of juvenile male mice

Exposing female house mice (Mus musculus) to male urinary scent accelerates their sexual development (Vandenbergh effect). Here, we tested whether exposing juvenile male mice to females’ urine similarly influences male growth and size of their sexual organs. We exposed three-week old male house mice to female urine or water (control) for ca. three months. We found that female-exposed males grew significantly faster and gained more body mass than controls, despite all males being reared on a controlled diet, but we detected no differences in males' muscle mass or sexual organs. In contrast, exposing juvenile males to male urine had no effect their growth. We tested whether the males' accelerated growth imposed functional trade-offs on males' immune resistance to an experimental infection. We challenged the same male subjects with an avirulent bacterial pathogen (Salmonella enterica), but found no evidence that faster growth impacted their bacterial clearance, body mass or survival during infection compared to controls. Our results provide the first evidence to our knowledge that juvenile male mice accelerate their growth when exposed to the urine of adult females, though we found no evidence that increased growth had negative trade-offs on immune resistance to infectious disease.

ECT9 condensates with ECT1 and regulates plant immunity.

Mounting an efficient defense against pathogens requires RNA binding proteins (RBPs) to regulate immune mRNAs transcription, splicing, export, translation, storage, and degradation. RBPs often have multiple family members, raising the question of how they coordinate to carry out diverse cellular functions. In this study, we demonstrate that EVOLUTIONARILY CONSERVED C-TERMINAL REGION 9 (ECT9), a member of the YTH protein family in Arabidopsis, can condensate with its homolog ECT1 to control immune responses. Among the 13 YTH family members screened, only ECT9 can form condensates that decrease after salicylic acid (SA) treatment. While ECT1 alone cannot form condensates, it can be recruited to ECT9 condensates in vivo and in vitro. Notably, the ect1/9 double mutant, but not the single mutant, exhibits heightened immune responses to the avirulent pathogen. Our findings suggest that co-condensation is a mechanism by which RBP family members confer redundant functions.

The intracellular immune receptor like gene SNC1 is an enhancer of effector-triggered immunity in Arabidopsis.

Plants contain many nucleotide-binding leucine-rich repeat (NLR) proteins that are postulated to function as intracellular immune receptors but do not yet have an identified function during plant-pathogen interactions. SUPPRESSOR OF NPR1-1, CONSTITUTIVE 1 (SNC1) is one such NLR protein of the Toll-interleukin 1 receptor (TIR) type, despite its well-characterized gain-of-function activity and its involvement in autoimmunity in Arabidopsis (Arabidopsis thaliana). Here, we investigated the role of SNC1 in natural plant-pathogen interactions and genetically tested the importance of the enzymatic activities of its TIR domain for its function. The SNC1 loss-of-function mutants were more susceptible to avirulent bacterial pathogen strains of Pseudomonas syringae containing specific effectors, especially under constant light growth condition. The mutants also had reduced defense gene expression induction and hypersensitive responses upon infection by avirulent pathogens under constant light growth condition. In addition, genetic and biochemical studies supported that the TIR enzymatic activity of SNC1 is required for its gain-of-function activity. In sum, our study uncovers the role of SNC1 as an amplifier of plant defense responses during natural plant-pathogen interactions and indicates its use of enzymatic activity and intermolecular interactions for triggering autoimmune responses.

The TGA Transcription Factors from Clade II Negatively Regulate the Salicylic Acid Accumulation in Arabidopsis.

Salicylic acid (SA) is a hormone that modulates plant defenses by inducing changes in gene expression. The mechanisms that control SA accumulation are essential for understanding the defensive process. TGA transcription factors from clade II in Arabidopsis, which include the proteins TGA2, TGA5, and TGA6, are known to be key positive mediators for the transcription of genes such as PR-1 that are induced by SA application. However, unexpectedly, stress conditions that induce SA accumulation, such as infection with the avirulent pathogen P. syringae DC3000/AvrRPM1 and UV-C irradiation, result in enhanced PR-1 induction in plants lacking the clade II TGAs (tga256 plants). Increased PR-1 induction was accompanied by enhanced isochorismate synthase-dependent SA production as well as the upregulation of several genes involved in the hormone’s accumulation. In response to avirulent P. syringae, PR-1 was previously shown to be controlled by both SA-dependent and -independent pathways. Therefore, the enhanced induction of PR-1 (and other defense genes) and accumulation of SA in the tga256 mutant plants is consistent with the clade II TGA factors providing negative feedback regulation of the SA-dependent and/or -independent pathways. Together, our results indicate that the TGA transcription factors from clade II negatively control SA accumulation under stress conditions that induce the hormone production. Our study describes a mechanism involving old actors playing new roles in regulating SA homeostasis under stress.

Host mixtures for plant disease control: Benefits from pathogen selection and immune priming.

Multiline and cultivar mixtures are highly effective methods for agroecological plant disease control. Priming‐induced cross protection, occurring when plants are challenged by avirulent pathogen genotypes and resulting in increased resistance to subsequent infection by virulent ones, is one critical key to their lasting performance against polymorphic pathogen populations. Strikingly, this mechanism was until recently absent from mathematical models aiming at designing optimal host mixtures. We developed an epidemiological model to explore the effect of host mixtures composed of variable numbers of single‐resistance cultivars on the equilibrium prevalence of the disease caused by pathogen populations polymorphic for virulence complexity. This model shows that a relatively large amount of resistance genes must be deployed to achieve low disease prevalence, as pathogen competition in mixtures tends to select for intermediate virulence complexity. By contrast, priming significantly reduces the number of plant genotypes needed to drop disease prevalence below an acceptable threshold. Given the limited availability of resistance genes in cultivars, this mechanism of plant immunity should be assessed when designing host mixtures.

Overexpression of NDR1 leads to pathogen resistance at elevated temperatures.

Summary Abiotic and biotic environments influence a myriad of plant‐related processes, including growth, development, and the establishment and maintenance of interaction(s) with microbes. In the case of the latter, elevated temperature has been shown to be a key factor that underpins host resistance and pathogen virulence.In this study, we elucidate a role for Arabidopsis NON‐RACE‐SPECIFIC DISEASE RESISTANCE1 (NDR1) by exploiting effector‐triggered immunity to define the regulation of plant host immunity in response to both pathogen infection and elevated temperature.We generated time‐series RNA sequencing data of WT Col‐0, an NDR1 overexpression line, and ndr1 and ics1‐2 mutant plants under elevated temperature. Not surprisingly, the NDR1‐overexpression line showed genotype‐specific gene expression changes related to defense response and immune system function.The results described herein support a role for NDR1 in maintaining cell signaling during simultaneous exposure to elevated temperature and avirulent pathogen stressors.

MTO1-RESPONDING DOWN 1 (MRD1) is a transcriptional target of OZF1 for promoting salicylic acid-mediated defense in Arabidopsis.

OZF1 promotes the transcription of MRD1, which is essential for SA-mediated defense against virulent and avirulent bacterial pathogens in Arabidopsis. Salicylic acid (SA) is critical for defense against biotrophic pathogens. A trans-activator protein NPR1 plays significant roles in SA-signaling. However, evidences suggest the existence of NPR1-independent pathways for SA signaling in plants. Previously, we reported Arabidopsis OXIDATION-RELATED ZN-FINGER PROTEIN1 (OZF1) as a positive regulator of NPR1-independent SA-signaling. However, the mechanism or components of OZF1-mediated SA signaling was not known. Through the analysis of differentially expressing genes, we report the identification of MTO1-RESPONDING DOWN 1 (MRD1) as a transcriptional target of OZF1. Expressions of MRD1 and its overlapping gene in Arabidopsis genome, HEI10 increase upon pathogen inoculation in an OZF1-dependent manner. Their mutants are susceptible to both virulent and avirulent bacterial pathogens and show compromised SA-mediated immunity. Overexpression of MRD1 but not the HEI10 rescues the loss-of-resistance phenotype of the ozf1 mutant. OZF1 physically associates at the MRD1 promoter area upon pathogen inoculation. Results altogether support that MRD1 is a transcriptional target of OZF1 for promoting SA-mediated defense in Arabidopsis.

Response of tomatoes primed by mycorrhizal colonization to virulent and avirulent bacterial pathogens

Most plants interact with arbuscular mycorrhizal fungi, which enhance disease resistance in the host plant. Because the effects of resistance against bacterial pathogens are poorly understood, we investigated the effects of mycorrhizal colonization on virulent and avirulent pathogens using phytopathological and molecular biology techniques. Tomato plants colonized by Gigaspora margarita acquired resistance not only against the fungal pathogen, Botrytis cinerea, but also against a virulent bacterial pathogen, Pseudomonas syringae pv. tomato DC3000 (Pst). In G. margarita-colonized tomato, salicylic acid (SA)- and jasmonic acid (JA)-related defense genes were expressed more rapidly and strongly compared to those in the control plants when challenged by Pst, indicating that the plant immunity system was primed by mycorrhizal colonization. Gene expression analysis indicated that primed tomato plants responded to the avirulent pathogen, Pseudomonas syringae pv. oryzae, more rapidly and strongly compared to the control plant, where the effect on the JA-mediated signals was stronger than in the case with Pst. We found that the resistance induced by mycorrhizal colonization was effective against both fungal and bacterial pathogens including virulent and avirulent pathogens. Moreover, the activation of both SA- and JA-mediated signaling pathways can be enhanced in the primed plant by mycorrhizal colonization.

Protection to Tomato Wilt Disease Conferred by the Nonpathogen Fusarium oxysporum Fo47 is More Effective Than that Conferred by Avirulent Strains.

Although the vascular pathogen Fusarium oxysporum is notorious for being the causal agent of Fusarium wilt disease, the vast majority of F. oxysporum strains are harmless soil and root colonizers. The latter F. oxysporum's are often endophytes colonizing roots intracellularly without negatively affecting plant fitness. Actually, most of them, like Fo47, are beneficial providing biological control to various root pathogens. Interestingly, also pathogenic F. oxysporum inoculated on a resistant host (i.e., avirulent F. oxysporum f. sp. lycopersici) can reduce susceptibility to virulent F. oxysporum strains via a mechanism called "cross protection." It has been hypothesized that cross protection is based on activation of a resistance protein of the host upon recognition of a cognate avirulence (Avr) protein of the pathogen. Currently, it is unknown whether the biocontrol activity of F. oxysporum endophytes utilizes similar mechanisms as cross protection conferred by avirulent pathogens, and whether both provide a quantitative similar level of protection. Here, we show that in tomato biocontrol activity of the Fo47 endophyte to the pathogen F. oxysporum f. sp. lycopersici is more effective than cross protection induced by avirulent F. oxysporum f. sp. lycopersici strains activating either I, I-2, or both resistance proteins upon recognition of Avr1 or the Avr2/Six5 pair, respectively. These findings imply that cross protection and biological control utilize different mechanisms to reduce susceptibility of the host to subsequent infections.

The Arabidopsis R2R3 MYB Transcription Factor MYB15 Is a Key Regulator of Lignin Biosynthesis in Effector-Triggered Immunity.

Lignin, a major component of the secondary cell wall, is important for plant growth and development. Moreover, lignin plays a pivotal role in plant innate immunity. Lignin is readily deposited upon pathogen infection and functions as a physical barrier that limits the spread of pathogens. In this study, we show that an Arabidopsis MYB transcription factor MYB15 is required for the activation of lignin biosynthesis genes such as PAL, C4H, 4CL, HCT, C3′H, COMT, and CAD, and consequently lignin formation during effector-triggered immune responses. Upon challenge with the avirulent bacterial pathogen Pst DC3000 (AvrRpm1), lignin deposition and disease resistance were reduced in myb15 mutant plants. Furthermore, whereas invading pathogens, together with hypersensitive cell death, were restricted to the infection site in wild-type leaves, they spread beyond the infected area in myb15 mutants. The exogenous supply of the lignin monomer coniferyl alcohol restored lignin production and rescued immune defects in myb15 plants. These results demonstrate that regulation at the transcriptional level is key to pathogen-induced lignification and that MYB15 plays a central role in this process.

Loss of proton/calcium exchange 1 results in the activation of plant defense and accelerated senescence in Arabidopsis

Cytosolic Ca2+ increases in response to many stimuli. CAX1 (H+/Ca2+ exchanger 1) maintains calcium homeostasis by transporting calcium from the cytosol to vacuoles. Here, we determined that the cax1 mutant exhibits enhanced resistance against both an avirulent biotrophic pathogen Pst-avrRpm1 (Pseudomonas syringae pv tomato DC3000 avrRpm1), and a necrotrophic pathogen, B. cinerea (Botrytis cinerea). The defense hormone SA (salicylic acid) and phytoalexin scopoletin, which fight against biotrophs and necrotrophs respectively, accumulated more in cax1 than wild-type. Moreover, the cax1 mutant exhibited early senescence after exogenous Ca2+ application. The accelerated senescence in the cax1 mutant was dependent on SID2 (salicylic acid induction deficient 2) but not on NPR1 (nonexpressor of pathogenesis-related genes1). Additionally, the introduction of CAX1 into the cax1 mutant resulted in phenotypes similar to that of wild-type in terms of Ca2+-conditioned senescence and Pst-avrRpm1 and B. cinerea infections. However, disruption of CAX3, the homolog of CAX1, did not produce an obvious phenotype. Moreover, exogenous Ca2+ application on plants resulted in increased resistance to both Pst-avrRpm1 and B. cinerea. Therefore, we conclude that the disruption of CAX1, but not CAX3, causes the activation of pathogen defense mechanisms, probably through the manipulation of calcium homeostasis or other signals.

Using Foldscope to Monitor Superoxide Production and Cell Death During Pathogen Infection in Arabidopsis Under Different Nitrogen Regimes.

Nitrogen nutrition plays a role in plant growth development and resistance against biotic and abiotic stress. During pathogen infection various signal molecules such as reactive oxygen species, calcium, reactive nitrogen species, salicylic acid, and ethylene plays an important role. The form of nitrogen nutrition such as nitrate or ammonium plays a role in production of these molecules. Under nitrate nutrition NO is predominant. The produced NO plays a role in reacting with superoxide to generate peroxynitrite to induce cell death during hypersensitive response elicited by avirulent pathogens. Excess of ROS is also detrimental to plants and NO plays a role in regulating ROS. Hence it is important to observe superoxide production during infection. By using an avirulent Pseudomonas syringae and Arabidopsis differential N nutrition we show superoxide production in leaves using a paper microscope called Foldscope, which can be applied as a simple microscope to observe objects. The data also compared with root system infected with pathogenic Fusarium oxysporum. Taken together here we show that Foldscope is a cost-effective and powerful technique to visualize superoxide and cell death in plants during infection.

Review of Concepts and Methods of Genetic Characterization and Breeding Wheat for Resistance to Rusts

Rust diseases are economically the most important constraints to global wheat production. Breeding for resistance to diseases involves the understanding of genetic systems of both the host and pathogen simultaneously. Knowledge of host-pathogen interaction is essential for breeding disease resistant crops, because both host and pathogen populations are genetically diverse. Resistance to rust diseases can be classified into two broad categories; seedling/ all stage resistance (ASR) and adult plant resistance (APR). All stage resistance genes confer resistance at all growth stages against avirulent pathogen isolates. However, ASR genes often succumb to evolution in pathogen populations, whereas APR genes are expressed at later growth stages of the plant, show uniform response to all races of the pathogen and do not encourage pathotypic evolution and the nature of resistance they offer is usually durable. Characterization of resistance to rusts involves gene postulation (multi-pathotype test), genetic analyses and identifying the number and location of the gene(s) conferring resistance. Chromosomal location and mapping genes have been done using cytogenetic analysis; however, since 1990s the advancement of molecular genetics and biotechnology enabled to replace the tedious, time, labor and resource consuming cytogenetic analyses method of locating genes using molecular mapping techniques.

Lignin-based barrier restricts pathogens to the infection site and confers resistance in plants.

Pathogenic bacteria invade plant tissues and proliferate in the extracellular space. Plants have evolved the immune system to recognize and limit the growth of pathogens. Despite substantial progress in the study of plant immunity, the mechanism by which plants limit pathogen growth remains unclear. Here, we show that lignin accumulates in Arabidopsis leaves in response to incompatible interactions with bacterial pathogens in a manner dependent on Casparian strip membrane domain protein (CASP)-like proteins (CASPLs). CASPs are known to be the organizers of the lignin-based Casparian strip, which functions as a diffusion barrier in roots. The spread of invading avirulent pathogens is prevented by spatial restriction, which is disturbed by defects in lignin deposition. Moreover, the motility of pathogenic bacteria is negatively affected bylignin accumulation. These results suggest that the lignin-deposited structure functions as a physical barrier similar to theCasparian strip, trapping pathogens and thereby terminating their growth.

Arabidopsis UBC13 differentially regulates two programmed cell death pathways in responses to pathogen and low-temperature stress.

UBC13 is required for Lys63-linked polyubiquitination and innate immune responses in mammals, but its functions in plant immunity remain to be defined. Here we used genetic and pathological methods to evaluate roles of Arabidopsis UBC13 in response to pathogens and environmental stresses. Loss of UBC13 failed to activate the expression of numerous cold-responsive genes and resulted in hypersensitivity to low-temperature stress, indicating that UBC13 is involved in plant response to low-temperature stress. Furthermore, the ubc13 mutant displayed low-temperature-induced and salicylic acid-dependent lesion mimic phenotypes. Unlike typical lesion mimic mutants, ubc13 did not enhance disease resistance against virulent bacterial and fungal pathogens, but diminished hypersensitive response and compromised effector-triggered immunity against avirulent bacterial pathogens. UBC13 differently regulates two types of programmed cell death in response to low temperature and pathogen. The lesion mimic phenotype in the ubc13 mutant is partially dependent on SNC1. UBC13 interacts with an F-box protein CPR1 that regulates the homeostasis of SNC1. However, the SNC1 protein level was not altered in the ubc13 mutant, implying that UBC13 is not involved in CPR1-regulated SNC1 protein degradation. Taken together, our results revealed that UBC13 is a key regulator in plant response to low temperature and pathogens.

Overexpression of phytoglobin in barley alters both compatible and incompatible interactions with the mildew pathogen Blumeria graminis

This study showed how barley plants can be shifted in their response to isolates of the mildew pathogen Blumeria graminis with different host adaptation by overexpression of the barley phytoglobin gene HvHb1. At early infection stages, plants overexpressing phytoglobin (GPHb1) showed less papilla formation and more hypersensitive response against both virulent and avirulent pathogen isolates compared to the wildtype (WT) plants. The shift was most pronounced in a wheat‐adapted isolate (B. graminis f. sp. tritici). At later infection stages, GPHb1 plants infected with a virulent pathogen isolate (A6) showed less leaf chlorosis compared to the WT plants, indicating delayed senescence. The chlorophyll level was significantly higher in A6‐infected GPHb1 plants 9 days after inoculation (dai) and the senescence indicators sphingosine‐1‐phosphate:ceramide ratio and phytol content confirmed delayed senescence. At 14 dai the percentage of fungal DNA was significantly higher on the GPHb1 plants than on WT plants, probably as a result of the delayed senescence. The results show that overexpression of phytoglobin (previously known as plant haemoglobin) can be an important tool to understand disease‐related stress effects in plants of agronomic importance and for understanding basic resistance mechanisms. Studying this process in more detail may provide insights into how to alleviate stress‐related senescence in plants.

TaMAPK4 Acts as a Positive Regulator in Defense of Wheat Stripe-Rust Infection.

Highly conserved mitogen-activated protein kinase (MAPK) cascades regulate numerous plant processes, including hormonal responses, stress, and innate immunity. In this research, TaMAPK4 was predicted to be a target of tae-miR164. We verified the binding and suppression of TaMAPK4 by co-expression in Nicotiana benthamiana. Moreover, we found TaMAPK4 was localized in the cytoplasm and nucleus using transient expression analyses. TaMAPK4 transcripts increased following salicylic acid (SA) treatment and when host plants were infected with an avirulent race of the stripe-rust pathogen. Silencing of TaMAPK4 by virus-induced gene silencing permitted increased colonization by the avirulent pathogen race. Detailed histological results showed increased Puccinia striiformis (Pst) hyphal length, hyphal branches, and infection uredinial size compared to the non-silenced control. SA accumulation and the transcript levels of TaPR1, TaPR2, and TaPR5 were significantly down-regulated in TaMAPK4 knockdown plants. Overall, these results suggest that TaMAPK4 plays an important role in signaling during the wheat-Pst interaction. These results present new insights into MAPK signaling in wheat defense to rust pathogen.