Cell Death In Tobacco Research Articles

Genome-Wide Identification and Characterization of Effector Candidates with Conserved Motif in Falciphora oryzae.

Microbes employ effectors to disrupt immune responses and promote host colonization. Conserved motifs including RXLR, LFLAK-HVLVxxP (CRN), Y/F/WxC, CFEM, LysM, Chitin-bind, DPBB_1 (PNPi), and Cutinase have been discovered to play crucial roles in the functioning of effectors in filamentous fungi. Nevertheless, little is known about effectors with conserved motifs in endophytes. This research aims to discover the effector genes with conserved motifs in the genome of rice endophyte Falciphora oryzae. SignalP identified a total of 622 secreted proteins, out of which 227 were predicted as effector candidates by EffectorP. By utilizing HMM features, we discovered a total of 169 effector candidates with conserved motifs and three novel motifs. Effector candidates containing LysM, CFEM, DPBB_1, Cutinase, and Chitin_bind domains were conserved across species. In the transient expression assay, it was observed that one CFEM and one LysM activated cell death in tobacco leaves. Moreover, two CFEM and one Chitin_bind inhibited cell death induced by Bax protein. At various points during the infection, the genes' expression levels were increased. These results will help to identify functional effector proteins involving omics methods using new bioinformatics tools, thus providing a basis for the study of symbiosis mechanisms.

An effector of Erysiphe necator translocates to chloroplasts and plasma membrane to suppress host immunity in grapevine.

The powdery mildew (Erysiphe necator) is a prevalent pathogen hampering grapevine growth in the vineyard. An arsenal of candidate secreted effector proteins (CSEPs) was encoded in the E. necator genome, but it is largely unclear what role CSEPs plays during the E. necator infection. In the present study, we identified a secreted effector CSEP080 of E. necator, which was located in plant chloroplasts and plasma membrane. Transient expressing CSEP080 promotes plant photosynthesis and inhibits INF1-induced cell death in tobacco leaves. We found that CSEP080 was a necessary effector for the E. necator pathogenicity, which interacted with grapevine chloroplast protein VviB6f (cytochrome b6-f complex iron-sulfur subunit), affecting plant photosynthesis. Transient silencing VviB6f increased the plant hydrogen peroxide production, and the plant resistance to powdery mildew. In addition, CSEP080 manipulated the VviPE (pectinesterase) to promote pectin degradation. Our results demonstrated the molecular mechanisms that an effector of E. necator translocates to host chloroplasts and plasma membrane, which suppresses with the grapevine immunity system by targeting the chloroplast protein VviB6f to suppress hydrogen peroxide accumulation and manipulating VviPE to promote pectin degradation.

A novel secreted protein FgHrip1 from Fusarium graminearum triggers immune responses in plants

Fusarium graminearum, the primary pathogenic fungus responsible for Fusarium head blight (FHB) in wheat, secretes abundant chemical compounds that interact with host plants. In this study, a secreted protein FgHrip1, isolated from the culture filtrate of F. graminearum, was found to induce typical cell death in tobacco. The FgHrip1 gene was then cloned and expressed in Escherichia coli. Further bioassay analysis showed that the recombinant FgHrip1 induced early defense induction events, such as reactive oxygen species (ROS) production, callose deposition, and up-regulation of defense-related genes in tobacco. Furthermore, FgHrip1 significantly enhanced immunity in tobacco seedlings against Pseudomonas syringae pv. tabaci 6605 (Pst. 6605) and tobacco mosaic virus (TMV). FgHrip1-treated wheat spikes also exhibited defense-related transcript accumulation and developed immunity against FHB infection. Whereas the expression of FgHrip1 was induced during the infection process, the deletion of the gene impaired the virulence of F. graminearum. Our results suggest that FgHrip1 triggers immunity and induces disease resistance in tobacco and wheat, thereby providing new insight into strategy for biocontrol of FHB.

A Novel Effector, FSE1, Regulates the Pathogenicity of Fusarium oxysporum f. sp. cubense Tropical Race 4 to Banana by Targeting the MYB Transcription Factor MaEFM-Like.

Phytopathogenic fungi secretes a range of effectors to manipulate plant defenses. Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4) is a soil-borne pathogen that causes destructive banana wilt disease. Understanding the molecular mechanisms behind Foc TR4 effectors and their regulation of pathogenicity is helpful for developing disease control strategies. In the present study, we identified a novel effector, Fusarium special effector 1 (FSE1), in Foc TR4. We constructed FSE1 knock-out and overexpression mutants and investigated the functions of this effector. In vitro assays revealed that FSE1 was not required for vegetative growth and conidiation of Foc TR4. However, inoculation analysis of banana plantlets demonstrated that knock-out of FSE1 increased the disease index, while overexpression of FSE1 decreased it. Microscope analysis suggested that FSE1 was distributed in the cytoplasm and nuclei of plant cells. Furthermore, we identified an MYB transcription factor, MaEFM-like, as the target of FSE1, and the two proteins physically interacted in the nuclei of plant cells. In addition, Transient expression of MaEFM-like induced cell death in tobacco leaves. Our findings suggest that FSE1 is involved in the pathogenicity of Foc TR4 by targeting MaEFM-like.

Functional Identification of Arthrinium phaeospermum Effectors Related to Bambusa pervariabilis × Dendrocalamopsis grandis Shoot Blight.

The shoot blight of Bambusa pervariabilis × Dendrocalamopsis grandis caused by Arthrinium phaeospermum made bamboo die in a large area, resulting in serious ecological and economic losses. Dual RNA-seq was used to sequence and analyze the transcriptome data of A. phaeospermum and B. pervariabilis × D. grandis in the four periods after the pathogen infected the host and to screen the candidate effectors of the pathogen related to the infection. After the identification of the effectors by the tobacco transient expression system, the functions of these effectors were verified by gene knockout. Fifty-three differentially expressed candidate effectors were obtained by differential gene expression analysis and effector prediction. Among them, the effectors ApCE12 and ApCE22 can cause programmed cell death in tobacco. The disease index of B. pervariabilis × D. grandis inoculated with mutant ΔApCE12 and mutant ΔApCE22 strains were 52.5% and 47.5%, respectively, which was significantly lower than that of the wild-type strains (80%), the ApCE12 complementary strain (77.5%), and the ApCE22 complementary strain (75%). The tolerance of the mutant ΔApCE12 and mutant ΔApCE22 strains to H2O2 and NaCl stress was significantly lower than that of the wild-type strain and the ApCE12 complementary and ApCE22 complementary strains, but there was no difference in their tolerance to Congo red. Therefore, this study shows that the effectors ApCE12 and ApCE22 play an important role in A. phaeospermum virulence and response to H2O2 and NaCl stress.

The N-terminus of a Fusarium graminearum-secreted protein enhances broad-spectrum disease resistance in plants.

Fusarium head blight is a destructive disease caused by Fusarium species. Little is known about the pathogenic molecular weapons of Fusarium graminearum. The gene encoding a small secreted protein, Fg02685, in F.graminearum was found to be upregulated during wheat head infection. Knockout mutation of Fg02685 reducedthe growth and development of Fusarium in wheat spikes. Transient expression of Fg02685 or recombinant protein led to plant cell death in a BAK1- and SOBIR1-independent system. Fg02685 was found to trigger plant basal immunity by increasing the deposition of callose, the accumulation of reactive oxygen species (ROS), and the expression of defence-related genes. The Fg02685 signal peptide was required for the plant's apoplast accumulation and induces cell death, indicating Fg02685 is a novel conserved pathogen-associated molecular pattern. Moreover, its homologues are widely distributed in oomycetes and fungal pathogens and induced cell death in tobacco. The conserved α-helical motif at the N-terminus was necessary for the induction of cell death. Moreover, a 32-amino-acid peptide, Fg02685 N-terminus peptide 32 (FgNP32), was essential for the induction of oxidative burst, callose deposition, and mitogen-activated protein kinase signal activation in plants. Prolonged exposure to FgNP32 enhanced the plant's resistance to Fusarium and Phytophthora. This study provides new approaches for an environment-friendly control strategy for crop diseases by applying plant immune inducers to strengthen broad-spectrum disease resistance in crops.

Novel Functional Analysis for Pathogenic Proteins of Bursaphelenchus xylophilus in Pine Seed Embryos Using a Virus Vector.

Pine wilt disease (PWD), which is caused by the pine wood nematode Bursaphelenchus xylophilus, is among the most serious tree diseases worldwide. PWD is thought to be initiated by sequential excessive hypersensitive responses to B. xylophilus. Previous studies have reported candidate pathogenic molecules inducing hypersensitive responses in pine trees susceptible to B. xylophilus. The functions of some of these molecules have been analyzed in model plants using transient overexpression; however, whether they can induce hypersensitive responses in natural host pines remains unclear due to the lack of a suitable functional analysis method. In this study, we established a novel functional analysis method for susceptible black pine (Pinus thunbergii) seed embryos using transient overexpression by the Apple latent spherical virus vector and investigated five secreted proteins of B. xylophilus causing cell death in tobacco to determine whether they induce hypersensitive responses in pine. We found that three of five molecules induced significantly higher expression in pathogenesis-related genes ( p < 0.05), indicating hypersensitive response in pine seed embryos compared with mock and green fluorescence protein controls. This result suggests that tobacco-based screening may detect false positives. This study is the first to analyze the function of pathogenic candidate molecules of B. xylophilus in natural host pines using exogenous gene expression, which is anticipated to be a powerful tool for investigating the PWD mechanism.

Cell death signalling is competitively but coordinately regulated by repressor-type and activator-type ethylene response factors in tobacco (Nicotiana tabacum) plants.

Ethylene response factors (ERFs) comprise one of the largest transcription factor families in many plant species. Tobacco (Nicotiana tabacum) ERF3 (NtERF3) and other ERF-associated amphiphilic repression (EAR) motif-containing ERFs are known to function as transcriptional repressors. NtERF3 and several repressor-type ERFs induce cell death in tobacco leaves and are also associated with a defence response against tobacco mosaic virus (TMV). We investigated whether transcriptional activator-type NtERFs function together with NtERF3 in the defence response against TMV infection by performing transient ectopic expression, together with gene expression, chromatin immunoprecipitation (ChIP) and promoter analyses. Transient overexpression of NtERF2 and NtERF4 induced cell death in tobacco leaves, albeit later than that induced by NtERF3. Fusion of the EAR motif to the C-terminal end of NtERF2 and NtERF4 abolished their cell death-inducing ability. The expression of NtERF2 and NtERF4 was upregulated at the early phase of N gene-triggered hypersensitive response (HR) against TMV infection. The cell death phenotype induced by overexpression of wild-type NtERF2 and NtERF4 was suppressed by co-expression of an EAR motif-deficient form of NtERF3. Furthermore, ChIP and promoter analyses suggested that NtERF2, NtERF3 and NtERF4 positively or negatively regulate the expression of NtERF3 by binding to its promoter region. Overall, our results revealed the cell death-inducing abilities of genes encoding activator-type NtERFs, including NtERF2 and NtERF4, suggesting that the HR-cell death signalling via the repressor-type NtERF3 is competitively but coordinately regulated by these NtERFs.

Plant Peroxisome-Targeting Effector MoPtep1 Is Required for the Virulence of Magnaporthe oryzae.

Rice blast caused by Magnaporthe oryzae is one of the most serious fungous diseases in rice. In the past decades, studies have reported that numerous M. oryzae effectors were secreted into plant cells to facilitate inoculation. Effectors target host proteins to assist the virulence of pathogens via the localization of specific organelles, such as the nucleus, endoplasmic reticulum, chloroplast, etc. However, studies on the pathogenesis of peroxisome-targeting effectors are still limited. In our previous study, we analyzed the subcellular localization of candidate effectors from M. oryzae using the agrobacterium-mediated transient expression system in tobacco and found that MoPtep1 (peroxisomes-targeted effector protein 1) localized in plant peroxisomes. Here, we proved that MoPtep1 was induced in the early stage of the M. oryzae infection and positively regulated the pathogenicity, while it did not affect the vegetative growth of mycelia. Subcellular localization results showed that MoPtep1 was localized in the plant peroxisomes with a signal peptide and a cupredoxin domain. Sequence analysis indicated that the homologous protein of MoPtep1 in plant-pathogenic fungi was evolutionarily conserved. Furthermore, MoPtep1 could suppress INF1-induced cell death in tobacco, and the targeting host proteins were identified using the Y2H system. Our results suggested that MoPtep1 is an important pathogenic effector in rice blast.

A Novel, Small Cysteine-Rich Effector, RsSCR10 in Rhizoctonia solani Is Sufficient to Trigger Plant Cell Death.

The necrotrophic phytopathogen Rhizoctonia solani (R. solani) is a fungus that causes disease in a wide range of plant species. Fungal genomes encode abundant, small cysteine-rich (SCR) secreted proteins, and the probable importance of these to pathogenesis has been highlighted in various pathogens. However, there are currently no reports of an R. solani SCR-secreted protein with evidential elicitor activity. In this study, the molecular function of 10 SCR-secreted protein genes from R. solani was explored by agroinfiltration into Nicotiana benthamiana (N. benthamiana) leaves, and a novel SCR protein RsSCR10 was identified that triggered cell death and oxidative burst in tobacco. RsSCR10 comprises 84 amino acids, including a signal peptide (SP) of 19 amino acids that is necessary for RsSCR10 to induce tobacco cell death. Elicitation of cell death by RsSCR10 was dependent on Hsp90 but not on RAR1, proving its effector activity. Two cysteine residues have important effects on the function of RsSCR10 in inducing cell death. Furthermore, RsSCR10 showed cross-interaction with five rice molecules, and the inferred functions of these rice proteins suggest they are instrumental in how the host copes with adversity. Overall, this study demonstrates that RsSCR10 is a potential effector that has a critical role in R. solani AG1 IA-host interactions.

Cooperative roles of introns 1 and 2 of tobacco resistance gene N in enhanced N transcript expression and antiviral defense responses

The tobacco virus resistance gene N contains four introns. Transient expression of transcripts from an N transgene containing these introns and driven by the native promoter in the presence of the elicitor of tobacco mosaic virus resulted in its increased expression. The requirement of the native promoter, the elicitor, or the individual introns for enhanced expression of N has not been fully studied. Here, we determined that 35S promoter-driven N transcript expression could be enhanced in the presence of the four introns regardless of the co-expression of the virus elicitor in tobacco. Function analyses using a series of N transgenes with different combination of introns revealed that the presence of intron 1 more so than intron 2 allowed higher accumulation of premature and mature N transcripts; however, both introns were important for not only enhanced gene expression but also for induction of cell death in tobacco and induced local resistance to spread of virus in Nicotiana benthamiana. Our findings indicate that introns 1 and 2 cooperatively contribute to N expression and virus resistance.

H2O2 signaling modulates Glycoprotein-1 induced programmed cell death in tobacco suspension cells

Glycoprotein (GP)-1 is a glycoprotein elicitor with antiviral activity found in Streptomyces kanasensis zx01. GP-1 can induce programmed cell death (PCD) in vitro; however, the underlying mechanism is unclear. In the present study, we demonstrated that GP-1 induced PCD in tobacco suspension cells, which was modulated by hydrogen peroxide (H2O2). GP-1 participated in and modulated biologically relevant signaling in plant cells. GP-1 induced tobacco cell death in a dose- and time-dependent manner; affected the expression of BRI1-associated receptor kinase 1 (BAK1) and the accumulation of salicylic acid (SA), which are related to PCD; and enzymatic activities and mitochondrial functions. In conclusion, GP-1-induced PCD in tobacco may be mediated by H2O2 which alters BAK1 and SA levels, as well as mitochondrial and gene function. This cell signal cascade played an important role in the process of GP-1 induced plant disease resistance.

Modulation of chloroplast components and defense responses during programmed cell death in tobacco infected with Pseudomonas syringae

We examined how tobacco plants coordinate chloroplast components and defense responses during Pseudomonas syringae pv. tomato (Pst) infection. Tobacco leaves infiltrated with Pst induced weak necrosis at 24 h post-infiltration (hpi) and severe necrosis at 48 hpi. Membrane damage, as shown by cellular leakage and malondialdehyde, and H2O2 production began to increase at 12 hpi and continuously increased at 24–72 hpi in Pst-infiltrated leaves. Pst infection resulted in decreases in light-harvesting chlorophyll-binding proteins (Lhc), Lhcb transcripts, electron transport rate, and Fv/Fm, indicating the impairment in structure and function of photosystem II. Photochemical quenching, qP, continuously decreased in Pst-infiltrated leaves at 24–48 hpi, whereas nonphotochemical quenching, NPQ, exhibited a 2-fold increase at 24 hpi and a decrease at 48 dpi. In response to Pst infection, chlorophyll began to decrease at 48 hpi, whereas levels of protoporphyrin IX (Proto IX), Mg-Proto IX, Mg-Proto methylester, and protochlorophyllide drastically decreased or disappeared as early as 24 hpi. Pst-infiltrated leaves greatly up-regulated the expression of ROS scavenging genes, Fe-SOD, APX, and CAT1, as well as defense-related genes, PII, PR1, PR2, PALa, and CHS1. Our study suggests that the modulation of photosynthetic components during pathogen infection, particularly in relation to the fast degradation of photosensitizing porphyrin intermediates and the increase in photoprotective NPQ, may contribute to attenuating cellular damage in the early stages of programmed cell death induced by Pst.

Haustoria - arsenals during the interaction between wheat and Puccinia striiformis f. sp. tritici.

SummaryAs an obligate parasite, Puccinia striiformis f. sp. tritici (Pst) forms haustoria to obtain nutrients from plant cells for development, and these structures are essential for pathogen survival. To better understand the contribution of haustoria to the interactions with the host plants, we isolated haustoria from susceptible wheat leaves infected with Pst race CYR31 and sequenced their transcriptome as well as those of urediospores and germ tubes, and compared the three transcriptomes. A total of 3524 up‐regulated genes were obtained from haustoria, of which 73 genes were related to thiamine biosynthesis, glycolysis and lipid metabolic processes. Silencing seven of the genes reduced the growth and development of Pst in wheat. More interestingly, 1197 haustorial secreted proteins (HASPs) were detected in haustoria, accounting for 34% of the total proteins, indicating that these HASPs play important roles in haustorium‐mediated pathogenic progression. Furthermore, 69 HASPs were able to suppress Bax‐triggered programmed cell death in tobacco. Additionally, 46 HASPs significantly reduced callose deposition in wheat using the type III secretion system. This study identified a large number of effectors through transcriptome sequencing, and the results revealed components of metabolic pathways that impact the growth and colonization of the pathogen and indicate essential functions of haustoria in the growth and pathogenicity of Pst.

Novel Cell Death-Inducing Elicitors from Phytophthora palmivora Promote Infection on Hevea brasiliensis.

Elicitors play an important role in plant and pathogen interactions. The discovery of new elicitors and their effects on plant defense responses is significant and challenging. In this study, we investigated novel elicitors from Phytophthora palmivora and their effects on plant defenses. A crude elicitor isolated by ethanol precipitation from culture filtrates of P. palmivora induced cell death in tobacco leaves. When tobacco leaves were infiltrated with this cell death-inducing elicitor, the accumulations of H2O2, salicylic acid (SA), scopoletin (Scp), and abscisic acid (ABA) were detected. Accumulations of SA, Scp, and ABA were also induced in rubber tree leaves. P. palmivora infection significantly increased in rubber tree leaves pretreated with the elicitor and cotreated with the elicitor and zoospores of P. palmivora. This elicitor can be described as compound elicitor because Fourier-transform infrared spectroscopy revealed that it consisted of both polysaccharide and protein. We also found that the cell death effect caused by this compound elicitor was completely neutralized by Proteinase K. The compound elicitor was composed of four fractions which were beta-glucan, high-molecular-weight glycoprotein, broad-molecular-weight glycoprotein and 42-kDa protein. Interestingly, the broad-molecular-weight glycoprotein caused the highest level of cell death in tobacco leaves, while the beta-glucan had no effect. The high-molecular-weight glycoprotein, broad-molecular-weight glycoprotein and 42-kDa protein fractions not only caused cell death in tobacco leaves but also induced high levels of SA accumulation. Furthermore, these three fractions clearly promoted P. palmivora infection of rubber tree leaves.

Sulphated Polysaccharide from Acanthophora spicifera Induced Hevea brasiliensis Defense Responses Against Phytophthora palmivora Infection.

Elicitors from seaweeds are considered an alternative stimulant of plant defenses against pathogenic infection. Finding new sources of elicitors and exploring their effects on plant defenses is a significant undertaking. In this study, we extracted crude polysaccharide (CPS) from Acanthophora spicifera (a red alga) and tested the effects of the compound on rubber tree (Hevea brasiliensis) defense responses. Accumulations of salicylic acid (SA) and scopoletin (Scp) were measured by HPLC. The expression of SA- and Jasmonic acid (JA)-responsive genes was analyzed by semi-qRT-PCR. Strong anion exchange chromatography and Fourier-transform infrared (FTIR) spectroscopy were used for purification and functional characterization of CPS, respectively. The extracted CPS enhanced rubber tree defenses against Phytophthora palmivora infection. It induced SA and Scp accumulations and SA-responsive gene expression, but suppressed JA-responsive gene expression. We successfully separated the non-sulphated polysaccharide (F1) from the sulphated polysaccharides (SPS). Both peaks of SPS (F2 and F3) were identified as lambda (λ)-carrageenan. The F3 fraction showed greater elicitor activity on tobacco leaves. It induced SA and Scp accumulations and peroxidase activity but suppressed catalase activity. Furthermore, the purified λ-carrageenan did not cause cell death in tobacco or rubber tree leaves. Therefore, the elicitor from A. spicifera could be an alternative plant stimulant.

Center Rot of Onion (Allium cepa) Caused by Pantoea ananatis Requires pepM, a Predicted Phosphonate-Related Gene

Pantoea ananatis, a cause of center rot of onion, is problematic in the United States and elsewhere. The bacterium lacks disease determinants common to most other bacterial pathogens of plants. A genomic island containing the gene pepM was detected within many onion-pathogenic strains of P. ananatis of diverse origins. The pepM gene of P. ananatis putatively encodes a protein that converts phosphoenolpyruvate to phosphonopyruvate, the first step in the biosynthesis of phosphonates and related molecules. This gene appears to be essential for center rot disease. Deletion of pepM rendered the mutant strain unable to cause lesions in leaves of growing onions and water-soaking of inoculated yellow onion bulbs. Furthermore, growth of the deletion mutant in onion leaves was significantly diminished compared with wild-type bacteria, and the mutant failed to cause cell death in tobacco. Complementation of the mutated strain with pepM restored the phenotype to wild-type capability. The pepM gene is the first pathogenicity factor identified that affects bacterial fitness as well as symptom development in both leaves and bulbs in a pathogen causing center rot of onion.

Reactive oxygen species mediate tapetal programmed cell death in tobacco and tomato

BackgroundHybrid vigor is highly valued in the agricultural industry. Male sterility is an important trait for crop breeding. Pollen development is under strict control of both gametophytic and sporophytic factors, and defects in this process can result in male sterility. Both in the dicot Arabidopsis and in the moncot rice, proper timing of programmed cell death (PCD) in the tapetum ensures pollen development. Dynamic ROS levels have been reported to control tapetal PCD, and thus pollen development, in Arabidopsis and rice. However, it was unclear whether it is evolutionarily conserved, as only those two distantly related species were studied.ResultsHere, we performed histological analyses of anther development of two economically important dicot species, tobacco and tomato. We identified the same ROS amplitude during anther development in these two species and found that dynamic ROS levels correlate with the initiation and progression of tapetal PCD. We further showed that manipulating ROS levels during anther development severely impaired pollen development, resulting in partial male sterility. Finally, real-time quantitative PCR showed that several tobacco and tomato RBOHs, encoding NADPH oxidases, are preferentially expressed in anthers.ConclusionThis study demonstrated evolutionarily conserved ROS amplitude during anther development by examining two commercially important crop species in the Solanaceae. Manipulating ROS amplitude through genetic interference of RBOHs therefore may provide a practical way to generate male sterile plants.

TaDIR1-2, a Wheat Ortholog of Lipid Transfer Protein AtDIR1 Contributes to Negative Regulation of Wheat Resistance against Puccinia striiformis f. sp. tritici.

Very few LTPs have been shown to act through plasma membrane receptors or to be involved in the hypersensitive response (HR). DIR1, a new type of plant LTP interacts with lipids in vitro, moves to distant tissues during systemic acquired resistance (SAR) and therefore is thought to be involved in long-distance signaling during SAR. However, the exact functions of DIR1 orthologs in cereal species under biotic and abiotic stresses have not been thoroughly defined. In this study, a novel wheat ortholog of the DIR1 gene, TaDIR1-2, was isolated from Suwon11, a Chinese cultivar of wheat and functionally characterized. Phylogenetic analysis indicated that TaDIR1-2 is clustered within the nsLTP-Type II group and shows a closer relationship with DIR1 orthologs from monocots than from eudicots. TaDIR1-2 was localized in the cytoplasm and the cell membrane of wheat mesophyll protoplast. Transcription of TaDIR1-2 was detected in wheat roots, stems and leaves. TaDIR1-2 transcript was significantly induced during the compatible interaction of wheat with the stripe rust pathogen, Puccinia striiformis f. sp. tritici (Pst). Treatments with salicylic acid (SA) and low temperature significantly up-regulated the expression of TaDIR1-2. Transient overexpression of TaDIR1-2 did not induce cell death or suppress Bax-induced cell death in tobacco leaves. Knocking down the expression of TaDIR1-2 through virus-induced gene silencing increased wheat resistance to Pst accompanied by HR, increased accumulation of H2O2 and SA, increased expression of TaPR1, TaPR2, TaPAL, and TaNOX, and decreased expression of two reactive oxygen species (ROS) scavenging genes TaCAT and TaSOD. Our results suggest that TaDIR1-2 acts as a negative regulator in wheat resistance to Pst by modulating ROS and/or SA-induced signaling.

TaMCA1, a regulator of cell death, is important for the interaction between wheat and Puccinia striiformis.

Metacaspase orthologs are conserved in fungi, protozoa and plants, however, their roles in plant disease resistance are largely unknown. In this study, we identified a Triticum aestivum metacaspase gene, TaMCA1, with three copies located on chromosomes 1A, 1B and 1D. The TaMCA1 protein contained typical structural features of type I metacaspases domains, including an N-terminal pro-domain. Transient expression analyses indicated that TaMCA1 was localized in cytosol and mitochondria. TaMCA1 exhibited no caspase-1 activity in vitro, but was able to inhibit cell death in tobacco and wheat leaves induced by the mouse Bax gene. In addition, the expression level of TaMCA1 was up-regulated following challenge with the Puccinia striiformis f. sp. tritici (Pst). Knockdown of TaMCA1 via virus-induced gene silencing (VIGS) enhanced plant disease resistance to Pst, and the accumulation of hydrogen peroxide (H2O2). Further study showed that TaMCA1 decreased yeast cell resistance similar to the function of yeast metacaspase, and there was no interaction between TaMCA1 and TaLSD1. Based on these combined results, we speculate that TaMCA1, a regulator of cell death, is important during the compatible interaction of wheat and Pst.