Analyzing autophagy in Magnaporthe oryzae

Magnaporthe oryzae is an important plant pathogenic fungus that greatly threatens the world's food security. Both genome-wide and individual gene studies have shown that the pathogenicity of the fungus is severely dependent on the intracellular autophagy process during appressoria development. This protocol discusses a systematic methodology to discover and monitor autophagy-related (ATG) genes in M. oryzae.     

Nonhost resistance to Magnaporthe oryzae in Arabidopsis thaliana

Rice blast, caused by Magnaporthe oryzae, is a devastating disease of rice (Oryza sativa). The mechanisms involved in resistance of rice to blast have been studied extensively and the rice—M. oryzae pathosystem has become a model for plant—microbe interaction studies. However, the mechanisms involved in nonhost resistance (NHR) of other plants to rice blast are still poorly understood. We have recently demonstrated that AGB1 and PMR5 contribute to PEN2-mediated preinvasion resistance to M. oryzae in Arabidopsis thaliana, suggesting a complex genetic network regulating the resistance. To determine whether other defense factors: RAR1, SGT1 and NHO1, affected the A. thaliana-M. oryzae interactions, double mutants were generated between pen2 and these defense-related mutants. All these double mutants exhibited a level of penetration resistance similar to that of the pen2 mutant, suggesting that none of these mutants significantly compromised resistance to M. oryzae in a pen2 background.Key words: nonhost resistance, PEN2, RAR1, SGT1, NHO1Plants face microbial attacks and have evolved innate immunity systems to defend against these threats. The initial step of the immunity signaling pathway is recognition of intra- or extracellular pathogen-derived molecules. Externally oriented transmembrane-type proteins containing leucine-rich repeat (LRR) domains detect extracellular molecules, whereas cytoplasmic sensors possess nucleotide-binding (NB) and LRR domains (NLR).^1,2 The LRR domain serves as a pattern-recognition receptor to detect pathogen-derived molecules or host proteins that are targeted by pathogen peptides that have entered the cell, effectors.³ NLR-type sensors are the substrates of a structurally and functionally conserved chaperone complex that consists of HEAT SHOCK PROTEIN 90 (HSP90) and its cochaperone SUPPRESSOR OF THE G2 ALLELE OF SKP1 (SGT1). REQUIRED FOR MLA12 RESISTANCE 1 (RAR1) regulated the HSP90-SGT1 complex, resulting in the stabilization of NLR proteins. Thus, SGT1 and RAR1 are required for the function of multiple and distinct R genes that encode NLR immune sensors in plants.⁴ Experiments in RAR1-silenced transgenic rice lines showed that RAR1 is not essential for Pib, which encodes an NLR against rice blast fungus.⁵ In contrast, basal resistance to normally virulent races of rice blast fungus or bacterial blight is significantly reduced in RAR1-silenced lines. This result is consistent with earlier reports that RAR1 is involved in basal resistance to virulent Pseudomonas bacteria in Arabidopsis or blast fungus in barley.^6,7 The requirement of SGT1 for immunity in plants is shown mostly by transient silencing of a number of NLR proteins.^8,9 In addition, SGT1 is also required for immune responses triggered by non-NLR-type sensors.¹⁰ This requirement indicates that either SGT1 function is not limited to the NLR sensors, or some unknown SGT1-dependent NLR proteins also operate downstream of non NLR-type sensors. Furthermore, SGT1 is involved in nonhost resistance, indicating that SGT1 may be a general factor of disease resistance.¹⁰ An Arabidopsis mutant, nho1 (nonhost resistance 1), has been isolated on which Pseudomonas syringae pv. phaseolicola grows and causes disease symptoms.^11,12 It is significant that this mutant is also compromised in R-gene-mediated resistance to P. syringae.¹¹ Although NHO1 is the flagellin-induced glycerol kinase, whose exact function in NHR remains elusive.^12,13 A possible explanation might be that altered plant glycerol pools either directly or indirectly affect nutrient availability for P. syringae. NHO1 is also required for resistance to the fungal pathogen Botrytis cinerea, indicating that NHO1 is not limited to bacterial resistance.¹² However, these contributions to NHR to M. oryzae in A. thaliana have not been understood.To determine whether these factors were necessary for the resistance to M. oryzae in A. thaliana, the following A. thaliana mutants were inoculated with M. oryzae and monitored by microscopy: rar1-21;¹⁴ edm1-1;¹⁵ nho1-1,¹¹ (all Col-0 background). All these mutants exhibited a level of penetration resistance similar to that of the wild-type plants (data not shown), suggesting that none of these mutants significantly compromised resistance to M. oryzae. We have recently shown that among the penetration (pen) mutants, only the pen2,¹⁶ mutant allowed increased penetration into epidermal cells by M. oryzae.¹⁷ Thus, double mutants were generated between pen2 and these mutants to determine whether these factors were necessary for the resistance to M. oryzae in a pen2 background: pen2 rar1-21; pen2 edm1-1; pen2 nho1-1. All these double mutants exhibited a level of penetration resistance similar to that of the pen2 mutant (Fig. 1), suggesting that none of these mutants significantly compromised resistance to M. oryzae in a pen2 background. This might indicate that NHR against M. oryzae may not be conferred by RAR1- and SGT1-dependent NLR immune sensors. Alternatively, since there has been no report that RAR1 is required for any known transmembrane sensors, such as FLS2, EFR or Xa21, RAR1- and SGT1-independent transmembrane-type immune sensors may be required for NHR against M. oryzae. Future studies will be required to reveal the genetic and mechanistic requirements for NHR in A. thaliana-M. oryzae interactions.Open in a separate windowFigure 1Double mutant analysis to evaluate the role of the defense related genes on resistance to Magnaporthe oryzae in Arabidopsis thaliana. The frequency of M. oryzae penetration on double mutants at 3 days post-inoculation was expressed as a percentage of total appressoria. Data were collected from six independent plants per line. A minimum of 100 infection sites was inspected per leaf. Results represent mean ± standard error of three independent experiments.     

Enzymatic detachment of spore germlings in Magnaporthe oryzae

Magnaporthe oryzae germlings tightly attach to the host surface by producing extracellular matrix (ECM) from germ tubes and appressoria, which are important for the early infection process. To understand the adhesion mechanisms of ECM during differentiation of infection structure, we evaluated the effects of various enzymes on M. oryzae germlings and the disease symptoms of the host plant, wheat. Treatment with β-mannosidase, collagenase N-2, collagenase S-1, or gelatinase B at 1-h postinoculation (hpi) resulted in germling detachment, although producing normal appressoria. Treatment with matrix metalloproteinases (MMPs) at 6 hpi also caused germling detachment. Furthermore, we confirmed by the inoculation tests and scanning electron microscopy that the germlings on the wheat plant were removed and did not manifest pathogenicity on treatment with MMPs. The most effective MMPs were crude collagenase, collagenase S-1, and gelatinase B, suggesting that the application of MMPs is promising for crop protection from fungal diseases by its detachment action.     

RNA silencing in the phytopathogenic fungus Magnaporthe oryzae

Systematic analysis of RNA silencing was carried out in the blast fungus Magnaporthe oryzae (formerly Magnaporthe grisea) using the enhanced green fluorescence protein (eGFP) gene as a model. To assess the ability of RNA species to induce RNA silencing in the fungus, plasmid constructs expressing sense, antisense, and hairpin RNAs were introduced into an eGFP-expressing transformant. The fluorescence of eGFP in the transformant was silenced much more efficiently by hairpin RNA of eGFP than by other RNA species. In the silenced transformants, the accumulation of eGFP mRNA was drastically reduced, but no methylation of the promoter or coding region was involved in it. In addition, we found small interfering RNAs (siRNAs) only in the silenced transformants. Interestingly, the siRNAs consisted of RNA molecules with at least three different sizes ranging from 19 to 23 nucleotides, and all of them contained both sense and antisense strands of the eGFP gene. To our knowledge, this is the first demonstration in which different molecular sizes of siRNAs have been found in filamentous fungi. Overall, these results indicate that RNA silencing operates in M. oryzae, which gives us a new tool for genome-wide gene analysis in this fungus.     

Penetration and Post-Penetration Resistance to Magnaporthe oryzae in Arabidopsis

The rate of entry of Magnaporthe oryzae into the Arabidopsis pen2 quintuple (pen2 NahG pmr5 agb1 mlo2) mutant was significantly higher than those into the pen2 quadruple (pen2 NahG pmr5 agb1 and pen2 NahG pmr5 mlo2) mutants. The lengths of the infection hyphae in the pen2 quintuple mutant were intermediate between the pen2 quadruple mutants. These results suggest that different genetic networks, consisting of PEN2, PMR5, AGB1, and MLO2, control penetration and post-penetration resistance to M. oryzae in Arabidopsis.     

A systematic analysis of T-DNA insertion events in Magnaporthe oryzae

We describe here the analysis of random T-DNA insertions that were generated as part of a large-scale insertional mutagenesis project for Magnaporthe oryzae. Chromosomal regions flanking T-DNA insertions were rescued by inverse PCR, sequenced and used to search the M. oryzae genome assembly. Among the 175 insertions for which at least one flank was rescued, 137 had integrated in single-copy regions of the genome, 17 were in repeated sequences, one had no match to the genome, and the remainder were unassigned due to illegitimate T-DNA integration events. These included in order of abundance: head-to-tail tandem insertions, right border excision failures, left border excision failures and insertion of one T-DNA into another. The left borders of the T-DNA were frequently truncated and inserted in sequences with micro-homology to the left terminus. By contrast the right borders were less prone to degradation and appeared to have been integrated in a homology-independent manner. Gross genome rearrangements rarely occurred when the T-DNAs integrated in single-copy regions, although most insertions did cause small deletions at the target site. Significant insertion bias was detected, with promoters receiving two times more T-DNA hits than expected, and open reading frames receiving three times fewer. In addition, we found that the distribution of T-DNA inserts among the M. oryzae chromosomes was not random. The implications of these findings with regard to saturation mutagenesis of the M. oryzae genome are discussed.     

稻瘟菌Magnaporthe oryzae P-ATPases基因家族分析

利用TCDB(Transporter Classification Database)网站数据库中的P-ATPases氨基酸序列对稻瘟菌全基因组表达序列(Coding Sequence,CDS)数据库进行搜索和分析,共发现23个P-ATPases基因,进化树分析表明这23个基因分属于4个家族和7个亚家族。构建了本地ESTs数据库,通过P-ATPases基因CDS序列与EST序列比对分析发现,在这些基因中有20个存在EST同源体,另外3个基因没有发现EST同源体,因此这20个基因是真实P-ATPases基因的可能性更高。运用MEME程序分析了这些P-ATPases蛋白结构域的基序,有6种基序在90%以上的基因氨基酸序列中出现,属保守基序。对这23个P-ATPases基因GC含量的分析表明,它们的平均GC含量在0.519-0.628之间,稍高于稻瘟菌整个基因组GC平均含量(0.516),同时这些基因内各区段GC含量变化不大,没有明显的梯度变化。本文结果为下一步深入研究稻瘟菌中P-ATPases基因家族的功能奠定了基础。     

ERECTA contributes to non-host resistance to Magnaporthe oryzae in Arabidopsis

ERECTA controls both developmental processes and disease resistance in Arabidopsis. We investigated the function of ERECTA in non-host resistance to Magnaporthe oryzae in Arabidopsis. In the pen2 er mutant, penetration resistance and post-penetration resistance to M. oryzae were compromised. These results suggest that ERECTA is involved in both penetration and post-penetration resistance to M. oryzae in Arabidopsis.     

Proteomic analysis of rice mutants susceptible to Magnaporthe oryzae

To identify genes involved in rice Pi5-mediated disease resistance to Magnaporthe oryzae, we compared the proteomes of the RIL260 rice strain carrying the Pi5 resistance gene with its susceptible mutants M5465 and M7023. Proteins were extracted from the leaf tissues of both RIL260 and the mutant lines at 0, 24, and 48 h after M. oryzae inoculation and separated by two-dimensional polyacrylamide gel electrophoresis (2-DE). Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis identified eight proteins that were differently expressed between the resistant and susceptible plants (three down- and five up-regulated proteins in the mutants). The down-regulated proteins included a triosephosphate isomerase (spot no. 2210), a 2,3-bisphosphoglycerate-independent phosphoglycerate mutase (no. 3611), and an unknown protein (no. 4505). In addition, the five up-regulated proteins in the mutants were predicted to be a fructokinase I (no. 313), a glutathione S-transferase (no. 2310), an atpB of chloroplast ATP synthase (no. 3616), an aminopeptidase N (no. 3724), and an unknown protein (no. 308). These results suggest that proteomic analysis of rice susceptible mutants is a useful method for identifying novel proteins involved in resistance to the M. oryzae pathogen.     

SOBIR1 contributes to non-host resistance to Magnaporthe oryzae in Arabidopsis

The rate of entry of Magnaporthe oryzae into Arabidopsis pen2 sobir1 plants was significantly higher than that into pen2 plants. The length of the infection hyphae in pen2 sobir1 plants was significantly longer than that in pen2 plants. These results suggest that SOBIR1 is involved in both penetration and post-penetration resistance to M. oryzae in Arabidopsis.     

Instability of the Magnaporthe oryzae avirulence gene AVR-Pita alters virulence

The avirulence gene AVR-Pita of Magnaporthe oryzae determines the efficacy of the resistance gene Pi-ta in rice. The structures of the AVR-Pita alleles in 39 US isolates of M. oryzae were analyzed using polymerase chain reaction. A series of allele-specific primers were developed from the AVR-Pita gene to examine the presence of AVR-Pita. Orthologous alleles of the AVR-Pita gene were amplified from avirulent isolates. Sequence analysis of five alleles revealed three introns at identical positions in the AVR-Pita gene. All five alleles were predicted to encode metalloprotease proteins highly similar to the AVR-Pita protein. In contrast, the same regions of the AVR-Pita alleles were not amplified in the most virulent isolates, and significant variations of DNA sequence at the AVR-Pita allele were verified by Southern blot analysis. A Pot3 transposon was identified in the DNA region encoding the putative protease motif of the AVR-Pita protein from a field isolate B2 collected from a Pi-ta-containing cultivar Banks. These findings show that transposons can contribute to instability of AVR-Pita and is one molecular mechanism for defeating resistance genes in rice cultivar Banks.     

Magnaporthe oryzae endopolygalacturonase homolog correlates with density-dependent conidial germination.

Endopolygalacturonases (endoPGs) of some phytopathogens are virulent factors for dicots. To investigate the function of the endoPG of Magnaporthe oryzae, a disruption mutant of MGG_08938, the homolog of endoPG found in the genome database of this fungus, was generated. The pathogenicity, mycelial growth, and appressorium formation of this mutant were comparable with those of the wild-type strain; however, the germination of conidia in a highly concentrated suspension of conidia was affected by the mutation. Whereas the germination of the wild-type strain was inhibited at high concentrations, this effect was canceled out by disruption by the endoPG homolog gene. The authors named the gene MDG1 (M. oryzae density-dependent germination), which delineates this new function in the fungus.     

A vacuolar glucoamylase,Sga1, participates in glycogen autophagy for proper asexual differentiation in Magnaporthe oryzae

Nutrient limitation acts as a trigger for the synthesis of glycogen, which serves as a carbon and energy reserve during starvation. Recently, we reported that an autophagy-deficient mutant (atg8Δ) shows severe reduction in aerial hyphal growth and conidiation in the rice-blast fungus Magnaporthe oryzae, and proposed that autophagy plays an important role in facilitating glycogen homeostasis to ensure proper asexual differentiation in Magnaporthe. Here, we identify and characterize a vacuolar glucoamylase function (Sga1) that hydrolyses glycogen to meet the energy requirements during asexual development in Magnaporthe. Loss of SGA1 resulted in significant reduction in conidiation compared to the wild-type Magnaporthe strain. More importantly, an sga1Δ atg8Δ double deletion mutant showed further reduction in conidiation compared to the atg8Δ mutant in Magnaporthe. Forced localization of GFP-Sga1 to the cytoplasm (through removal of the predicted signal peptide) led to increased conidiation in wild type and the sga1Δ, but more interestingly, significantly restored conidiation in the atg8Δ mutant. Our results indicate that autophagy and Sga1 act cooperatively in vacuolar glycogen breakdown, which is essential for conidia formation but dispensable for pathogenicity in Magnaporthe.     

Organization of chromosome ends in the rice blast fungus, Magnaporthe oryzae

Eukaryotic pathogens of humans often evade the immune system by switching the expression of surface proteins encoded by subtelomeric gene families. To determine if plant pathogenic fungi use a similar mechanism to avoid host defenses, we sequenced the 14 chromosome ends of the rice blast pathogen, Magnaporthe oryzae. One telomere is directly joined to ribosomal RNA-encoding genes, at the end of the ~2 Mb rDNA array. Two are attached to chromosome-unique sequences, and the remainder adjoin a distinct subtelomere region, consisting of a telomere-linked RecQ-helicase (TLH) gene flanked by several blocks of tandem repeats. Unlike other microbes, M.oryzae exhibits very little gene amplification in the subtelomere regions—out of 261 predicted genes found within 100 kb of the telomeres, only four were present at more than one chromosome end. Therefore, it seems unlikely that M.oryzae uses switching mechanisms to evade host defenses. Instead, the M.oryzae telomeres have undergone frequent terminal truncation, and there is evidence of extensive ectopic recombination among transposons in these regions. We propose that the M.oryzae chromosome termini play more subtle roles in host adaptation by promoting the loss of terminally-positioned genes that tend to trigger host defenses.     

Novel mating type-dependent transcripts at the mating type locus in Magnaporthe oryzae

The mating type locus (MAT1) of Magnaporthe oryzae has similar structural organization to MAT in other ascomycetes and encodes the mating type genes MAT1-1-1 with an alpha-box motif and MAT1-2-1 with an HMG-box motif in the MAT1-1 and MAT1-2 idiomorphs, respectively. Sequence and expression analyses of the MAT1 locus indicated a second open reading frame (ORF), MAT1-1-2, in the MAT1-1 idiomorph, and novel mating-type dependent ORFs (MAT1-1-3 and MAT1-2-2) at the locus. The MAT1-1-3 ORF initiated within the MAT1-1 idiomorph while the MAT1-2-2 ORF initiated at the border of the MAT1-2 idiomorph with both ORFs sharing most of their reading frames in the MAT1 flanking region. This suggests that the encoded proteins (MAT1-1-3 and MAT1-2-2) should be similar in their primary structures but can be distinguished by distinct N-termini with amino acids of 1 and 32, respectively, in each mating type. A CT dinucleotide repeat, (CT)n, present in the upstream region of MAT1-1-3, was polymorphic among the isolates.