MAT Loci Play Crucial Roles in Sexual Development but Are Dispensable for Asexual Reproduction and Pathogenicity in Rice Blast Fungus Magnaporthe oryzae.

Magnaporthe oryzae, a fungal pathogen that causes rice blast, which is the most destructive disease of rice worldwide, has the potential to perform both asexual and sexual reproduction. MAT loci, consisting of MAT genes, were deemed to determine the mating types of M. oryzae strains. However, investigation was rarely performed on the development and molecular mechanisms of the sexual reproduction of the fungus. In the present work, we analyzed the roles of two MAT loci and five individual MAT genes in the sex determination, sexual development and pathogenicity of M. oryzae. Both of the MAT1-1 and MAT1-2 loci are required for sex determination and the development of sexual structures. MAT1-1-1, MAT1-1-3 and MAT1-2-1 genes are crucial for the formation of perithecium. MAT1-1-2 impacts the generation of asci and ascospores, while MAT1-2-2 is dispensable for sexual development. A GFP fusion experiment indicated that the protein of MAT1-1-3 is distributed in the nucleus. However, all of the MAT loci or MAT genes are dispensable for vegetative growth, asexual reproduction, pathogenicity and pathogenicity-related developments of the fungus, suggesting that sexual reproduction is regulated relatively independently in the development of the fungus. The data and methods of this work may be helpful to further understand the life cycle and the variation of the fungus.

Pex13 and Pex14, the key components of the peroxisomal docking complex, are required for peroxisome formation, host infection and pathogenicity-related morphogenesis in Magnaporthe oryzae.

Peroxisomes are ubiquitous organelles in eukaryotic cells that fulfill multiple important metabolisms. Pex13 and Pex14 are key components of the peroxisomal docking complex in yeasts and mammals. In the present work, we functionally characterized the homologues of Pex13 and Pex14 (Mopex13 and Mopex14) in the rice blast fungus Magnaporthe oryzae. Mopex13 and Mopex14 were peroxisomal membrane distributed and were both essential for the maintenance of Mopex14/17 on the peroxisomal membrane. Mopex13 and Mopex14 interacted with each other, and with Mopex14/17 and peroxisomal matrix protein receptors. Disruption of Mopex13 and Mopex14 resulted in a cytoplasmic distribution of peroxisomal matrix proteins and the Woronin body protein Hex1. In the ultrastructure of Δmopex13 and Δmopex14 cells, peroxisomes were detected on fewer occasions, and the Woronin bodies and related structures were dramatically affected. The Δmopex13 and Δmopex14 mutants were reduced in vegetative growth, conidial generation and mycelial melanization, in addition, Δmopex13 showed reduced conidial germination and appressorial formation and abnomal appressorial morphology. Both Δmopex13 and Δmopex14 were deficient in appressorial turgor and nonpathogenic to their hosts. The infection failures in Δmopex13 and Δmopex14 were also due to their reduced ability to degrade fatty acids and to endure reactive oxygen species and cell wall-disrupting compounds. Additionally, Mopex13 and Mopex14 were required for the sexual reproduction of the fungus. These data indicate that Mopex13 and Mopex14, as key components of the peroxisomal docking complex, are indispensable for peroxisomal biogenesis, fungal development and pathogenicity in the rice blast fungus.

One of Three Pex11 Family Members Is Required for Peroxisomal Proliferation and Full Virulence of the Rice Blast Fungus Magnaporthe oryzae.

Peroxisomes play important roles in metabolisms of eukaryotes and infection of plant fungal pathogens. These organelles proliferate by de novo formation or division in response to environmental stimulation. Although the assembly of peroxisomes was documented in fungal pathogens, their division and its relationship to pathogenicity remain obscure. In present work, we analyzed the roles of three Pex11 family members in peroxisomal division and pathogenicity of the rice blast fungus Magnaporthe oryzae. Deletion of MoPEX11A led to fewer but enlarged peroxisomes, and impaired the separation of Woronin bodies from peroxisomes, while deletion of MoPEX11B or MoPEX11C put no evident impacts to peroxisomal profiles. MoPEX11A mutant exhibited typical peroxisome related defects, delayed conidial germination and appressoria formation, and decreased appressorial turgor and host penetration. As a result, the virulence of MoPEX11A mutant was greatly reduced. Deletion of MoPEX11B and MoPEX11C did not alter the virulence of the fungus. Further, double or triple deletions of the three genes were unable to enhance the virulence decrease in MoPEX11A mutant. Our data indicated that MoPEX11A is the main factor modulating peroxisomal division and is required for full virulence of the fungus.

[Bioinformatic research of the family of PEX11, peroxisome proliferous factor in fungus].

The family members of PEX11 are key factors involved in regulation of peroxisome proliferation. Sixty-six PEX11p candidates of PEX11 gene family from 26 representative fungal species were obtained and analyzed by bioinformatic strategies. In most filamentous fungi, 2 or 3 potential PEX11ps were found, in contrast with 1 or 2 in yeast species. Compared with other fungal species, the Ascomycetes tend to have more PEX11ps, and even 5 in several individuals. The data of phylogenetic analysis and protein structure indicated that all of the PEX11ps were divided into 3 groups: I, II, and III. The members of group I and group III existed in most species, while those in group II were found only in Pezizomycotina. By MEME analysis, 5-6 conserved motifs were found in each PEX11ps. Among them,motif 8 in C-terminal had the most conservation, indicating that this motif probably plays a key role in maintaining the proper function of PEX11p.

[Identification and classification of rice leaf blast based on multi-spectral imaging sensor].

Site-specific variable pesticide application is one of the major precision crop production management operations. Rice blast is a severe threat for rice production. Traditional chemistry methods can do the accurate crop disease identification, however they are time-consuming, require being executed by professionals and are of high cost. Crop disease identification and classification by human sight need special crop protection knowledge, and is low efficient. To obtain fast, reliable, accurate rice blast disease information is essential for achieving effective site-specific pesticide applications and crop management. The present paper describes a multi-spectral leaf blast identification and classification image sensor, which uses three channels of crop leaf and canopy images. The objective of this work was to develop and evaluate an algorithm under simplified lighting conditions for identifying damaged rice plants by the leaf blast using digital color images. Based on the results obtained from this study, the seed blast identification accuracy can be achieved at 95%, and the leaf blast identification accuracy can be achieved at 90% during the rice growing season. Thus it can be concluded that multi-spectral camera can provide sufficient information to perform reasonable rice leaf blast estimation.

Fluorescent co-localization of PTS1 and PTS2 and its application in analysis of the gene function and the peroxisomal dynamic in Magnaporthe oryzae.

The peroxisomal matrix proteins involved in many important biological metabolism pathways in eukaryotic cells are encoded by nucleal genes, synthesized in the cytoplasm and then transported into the organelles. Targeting and import of these proteins depend on their two peroxisomal targeting signals (PTS1 and PTS2) in sequence as we have known so far. The vectors of the fluorescent fusions with PTS, i.e., green fluorescence protein (GFP)-PTS1, GFP-PTS2 and red fluorescence protein (RFP)-PTS1, were constructed and introduced into Magnaporthe oryzae Guy11 cells. Transformants containing these fusions emitted fluorescence in a punctate pattern, and the locations of the red and green fluorescence overlapped exactly in RFP-PTS1 and GFP-PTS2 co-transformed strains. These data indicated that both PTS1 and PTS2 fusions were imported into peroxisomes. A probable higher efficiency of PTS1 machinery was revealed by comparing the fluorescence backgrounds in GFP-PTS1 and GFP-PTS2 transformants. By introducing both RFP-PTS1 and GFP-PTS2 into Deltamgpex6 mutants, the involvement of MGPEX6 gene in both PTS1 and PTS2 pathways was proved. In addition, using these transformants, the inducement of peroxisomes and the dynamic of peroxisomal number during the pre-penetration processes were investigated as well. In summary, by the localization and co-localization of PTS1 and PTS2, we provided a useful tool to evaluate the biological roles of the peroxisomes and the related genes.

[Strategies of targeted gene replacement in filamentous fungi].

Targeted gene replacement (TGR) is an important technique for gene function analysis. With the development of genome sequencing and transformation, TGR has been applied widely to filamentous fungi, and many new systems and approaches have been established. In this paper, strategies involved in TGR in filamentous fungi were reviewed, including transformation systems, targeting vector construction, mutant selection and so on. Compared with TGR, RNAi and other techniques of gene function analysis were introduced and reviewed as well.

Isolation and characterization of defense response genes involved in neck blast resistance of rice.

Two cDNA libraries enriched for transcripts differentially expressed in plants of two rice lines with similar genetic backgrounds and same leaf blast resistance but different responses to neck blast using suppression subtractive hybridization (SSH). After differential screening and sequence analysis of the selected clones, 90 unique cDNA clones were found, of which 74 clones were with known functions according to the putative functions of their homologous genes in the database. They may be involved in pathogen response, signal transduction, transcription, etc. Expression differences of 17 out of the 26 selected cDNA clones in resistant and susceptible lines were confirmed by RT-PCR. Expression profilings of the 26 cDNA clones at the early stages after inoculation were also revealed by RT-PCR. This is the first report on the rice neck blast resistance at mRNA level and will facilitate the further study of genetic mechanism of neck blast resistance.

Genetic dissections of partial resistances to leaf and neck blast in rice (Oryza sativa L.).

In a recombinant inbred line (RIL) population of indica rice, two subpopulations composed of susceptible lines were selected for mapping of partial resistance to leaf blast with two isolates of the pathogen. A third subpopulation composed of susceptible lines with similar heading time was used for mapping of partial resistance to neck blast with a third isolate. The traits measured for partial resistance included diseased leaf area (DLA), lesion size (LS) and lesion number (LN) for leaf blast and lesion length (LL) and conidium amount (CA) for neck blast. A linkage map consisting of 168 DNA markers was constructed by using the whole RIL population. Quantitative trait loci (QTLs) conditioning these traits were determined at one-locus and two-locus levels. Eleven main-effect QTLs and 28 digenic interactions were detected by QTLMapper 1.01 b. Only three QTLs showing main effects were also involved in digenic interactions for the same trait. General contributions of epistatic QTLs of each trait ranged from 16.0% to 51.7%, while those of main-effect QTLs of each trait ranged from 4.7% to 38.8%. The general contributions of main-effect QTLs of most traits were smaller than those of epistatic QTLs, confirming the importance of epistasis as the genetic basis for complex traits. The general contributions of the main and epistatic effects of all QTLs detected for the two traits LL and CA of the partial resistance to neck blast reached 70.6% and 82.6% respectively, which obviously represented a major part of the genetic basis controlling partial resistance to neck blast. The results indicated the necessity for partial resistance mapping to use susceptible subpopulations where the interference of major resistance genes is avoided.

[Analysis of gene expression profiles during host-Magnaporthe grisea interactions in a pair of near isogenic lines of rice].

A pair of near isogenic lines G205 and G71 were selected from recombinant inbred lines (RIL) of Zhong156 x Gumei2. On the resistance locus Pi-25(t), G205 had the resistant allele that was from Gumei 2 while G71 had the susceptible allele that was from Zhong156. For the genetic background, different alleles were detected on only 24 loci out of the 672 RFLP or SSLP loci surveyed. The expression profiles of G205 and G71 in response to Magnaporthe grisea were investigated using cDNA microarray containing 2200 Expression Sequence Tags (ESTs). The leaves were inoculated with the pathogen for 12 hours at 4-leaf stage and 998 genes were identified in total. Three genes were up-regulated significantly by the fungus in G205 only. The functions of two genes were known but that of the third gene were unknown. The two genes encoded casein kinase II alpha subunit and retrotransponson TOS17 insertion element respectively. Other thirty-five genes had similar expression patterns between NILs. Among them, 17 genes were up-regulated while 18 genes were down-regulated by the inoculation. The functions of 33 out of the 35 genes were known. BLAST analysis showed that all thirty-five. BLAST analysis showed that all thirty-five genes with known functions were relative to defense reactions, signal transduction, stress response, photosynthesis and sugar metabolism. Northern blot confirmed that four of five differentially displayed genes randomly selected had the same expression patterns as those detected in cDNA microarray. Two of them were up-regulated genes encoding casein kinase II alpha subunit and glycine-rich protein (Grp), and the other two down-regulated genes encoding nitrilase-associated protein and 18S small subnit ribosomal RNA gene respectively. Northern blot also revealed that the expression of Grp was consistently up-regulated from 0 to 36 h after the inoculation of the fungus. These results showed that cDNA microarray was a useful tool to study the molecular mechanisms of disease resistance in plants.