Abstract: Methods for silencing genes in Phytophthora transformants have been demonstrated previously, but wide variation in effectiveness was reported in different studies. To optimize this important tool for functional genomics, we compared the abilities of sense, antisense, and hairpin transgenes introduced by protoplast, electroporation, and bombardment methods to silence the inf1 elicitin gene in Phytophthora infestans. A hairpin construct induced silencing three times more often than sense or antisense vectors, and protoplast transformation twice as much as electroporation. Using hairpins introduced into protoplasts, 61% of strains were silenced, and transgene copy number was positively correlated with silencing. The utility of bombardment was reduced by the occurrence of heterokaryons containing silenced and non-silenced nuclei, but silenced strains were obtainable from about 20% of primary transformants by single-nuclear purification. Most inf1-deficient strains were fully silenced, however some exhibited partial suppression. These produced inf1-derived RNAs of about 21-nt which correspond to both the sense and antisense strands of inf1, implicating an RNAi-like mechanism in silencing.
Abstract: The internal transcribed spacer (ITS) region (ITS1, ITS2 and 5.8S rDNA) of the nuclear ribosomal DNA (nrDNA) was amplified via the PCR method in seventeen different isolates of Phytophthora sojae using the common primers of the ITS of fungi. Around 800 bp-1,000 bp fragments were obtained based on the DL2000 marker and the sequences of the PCR products were tested. Taking isolate USA as outgroup, the phylogenetic tree was constructed by means of maximum parsimony analysis, and the genetic evolution among isolates was analyzed. The results showed that there is a great difference between the base constitution of ITS1 and ITS2 among various isolates. The seventeen isolates are classified into three groups, and the isolates from the same region belong to the same group, which shows the variation in geography.
Abstract: The molecular mechanisms and the defense responses associated with partial resistance to Phytophthora sojae in soybean are unknown. In this study, we examined correlations between the expression of defense genes with partial resistance. First, to determine whether constitutive levels of expression of defense-related genes correlated with partial resistance to P. sojae, northern blot analysis of seven defense-related genes in 14 cultivars with low, moderate and high levels of partial resistance was performed. Pearson's correlations between mean lesion length and mean constitutive mRNA signals for defense-related genes showed no significant association to partial resistance to P. sojae. These results suggested that mechanisms linked to defense-related mRNA levels expressed during infection might better explain variations in partial resistance to P. sojae in soybean. Second, accumulation of four defense-related transcripts during infection was monitored in a spatial, time-course infection assay with two soybean cultivars, Conrad (high level of partial resistance) and OX 20-8 (Rps1a, low level of partial resistance). mRNA was isolated for Northern blot analysis from root sections harvested below, at, and above the inoculation site at 0, 6, 12, 24, 48 and 72 h after inoculation (hai) with P. sojae. P. sojae and soybean actin cDNAs were used as probes in the infected root sections to estimate relative proportions of RNA. Differential mRNA accumulation patterns for both soybean and P. sojae actin following P. sojae colonization in the three root sections of Conrad and OX 20-8 suggested that effective lesion-limiting mechanisms occurred primarily in the upper root section. Transcript levels for PR1a, matrix metalloproteinase (MMP) and basic peroxidase (IPER) at the inoculation site; and IPER above the inoculation site at 72 hai were significantly higher in Conrad with higher levels of partial resistance. Our results suggest that defense responses associated with accumulation of PR1a, MMP, IPER and β-1,3-endoglucanase (EGL) mRNAs may contribute to the partial resistance response to P. sojae in soybean.
Abstract: The effects of the glucan elicitor (WGE) from the cell wall of Phytophthora sojae on the transcriptional upregulation of genes representing five families of pathogenesis-related (PR) proteins have been investigated in soybean under wound and minimal-wound conditions. Although each has its own expression pattern, these PR protein genes can be broadly categorized into two groups. Induced expression of genes for PR-1a and PR-10 is predominantly limited to cells proximal to the point of treatment, while that of genes for the elicitor-releasing endo-glucanase (GLU, a PR-2), a WIN-like protein (WIN, a PR-4) and a Kunitz trypsin inhibitor (KTI, a PR-6) is seen in proximal, near-proximal and distal cells. GLU is the only family that shows weak though detectable constitutive (basal) gene expression. Wounding induces only barely detectable to weak expression of genes for PR-1a and KTI and somewhat stronger expression of those for GLU, WIN and PR-10. PR-1a gene expression is essentially induced only by WGE; similarly, that for KTI is greatly enhanced by WGE. While gene expression for PR-10 is induced by wounding, it is also strongly upregulated by WGE in a minimal wound background, suggesting a possibly independent effect of WGE. Finally, WGE also enhances the speed and magnitude of expression of GLU and WIN genes in all cell zones. Methyl jasmonate induces strong gene expression for KTI and the ethylene precursor, ACC, and jasmonic acid (JA) enhance that of GLU. The results are discussed in the context of the recently observed WGE- and JA-induced distal defense potentiation in soybean.
Abstract: The filamentous fungal genetics community has enthusiastically embraced the utilization of genomics technologies to resolve long-standing issues in fungal biology. For example, such technologies have been proposed to study the mechanics of tip growth, photoreception, gene silencing, the molecular basis of
conidiation, the pathway leading to sexual reproduction, and mechanisms of pathogenesis. These studies have provided a refreshing change of pace in research on filamentous fungi, which has lagged behind that on other eukaryotes in the exploitation of genome-wide methodologies. Despite the late start, several fungal genome sequencing projects are underway. The resulting databases will allow the comprehensive analysis of developmental processes that are characteristic of fungi, including the molecular nature of pathogenicity. DNA databases underpin analyses of the fungal transcriptome, proteome, and metabolome. This combined informationwill contribute to our basic understanding of not only the mechanics of infection but also the evolution of pathogenicity.
Abstract: Late blight caused by Phytophthora infestuns is the most serious disease of tomato production in China. Studies on the genetics of resistance and identification of molecular markers are very useful for breeding late blight resistant varieties. The objective of this paper was to study the inheritance of late blight resistance and identify simple sequence repeat (SSR) markers associated with resistance allele in tomato (Lycopersicon esculenturn Mill). The results came from an F2 progeny of 241 plants derived from a cross between 5# inbred line that is susceptible to late blight and a resistant accession CLN2037E. The late blight responses of F2 plants were tested by artificially inoculation of detached-leaflets in plate and natural infection assayed under greenhouse conditions. Both methods showed that the resistance is dominant and inherited as monogenic trait. Genetic mapping and linkage analysis showed that the late blight resistance gene Ph-ROL was located on chromosome 9 with a genetic distance of 5.7 cM to the SSR marker TOM236.
Abstract: A total of 1000 expressed sequence tags (ESTs) corresponding to 760 unique sequence sets were identified using random sequencing of clones from a cDNA library constructed from mycelial RNA of Phytophthora infestans. A number of software programs, represented by a relational database and an analysis pipeline, were developed for the automated analysis and storage of the EST sequence data. A set of 419 nonredundant sequences, which correspond to a total of 632 ESTs (63.2%), were identified as showing significant matches to sequences deposited in public databases. A putative cellular identity and role was assigned to all 419 sequences. All major functional categories were represented by at least several ESTs. Four novel cDNAs containing sequences related to elicitins, a family of structurally related proteins that induce the hypersensitive response and condition avirulence of P. infestans on Nicotiana plants, were among the most notable genes identified. Two of these elicitin-like cDNAs were among the most abundant cDNAs examined. The set also contained several ESTs with high sequence similarity to unique plant genes. Copyright 1999 Academic Press.
Abstract: To elucidate the molecular events of potato quantitative resistance to Phytophthora infestans, we performed a comprehensive transcriptional analysis using cDNA microarrays, containing 1009 ESTs from a subtractive library. Leaves of a moderately resistant potato clone were inoculated with P. infestans and sampled at nine time points ranging from 2 to 72 h after inoculation. A total of 348 P. infestans-responsive genes were identified. These functional genes are mostly related to metabolism, plant defense, signaling and transcription regulation, involving the whole process of plant defense response to pathogens. Based on the general expression patterns of these genes at different time points, we discriminated distinct stages of potato defense against P. infestans and revealed genes participating in each stage. To further understand the dynamics of P. infestans-induced gene expression, hierarchical clustering was used to illustrate their various expression profiles during the time course, including early, mid and late gene induction as well as early gene repression. Interestingly, some genes involved in the hypersensitive response were also identified, suggesting that a same or similar defense system may exist in both race-specific and race-nonspecific resistances. In addition, 114 novel genes with unknown functions were isolated.
Abstract:Gibberella zeae is a broad host range pathogen that infects many crop plants, including wheat and barley, and causes head blight and rot diseases throughout the world. To better understand fungal development and pathogenicity, we have generated 7996 ESTs from three cDNA libraries. Two libraries were generated from carbon-(C-) and nitrogen- (N-) starved mycelia and one library was generated from cultures of maturing perithecia (P). In other fungal pathogens, starvation conditions have been shown to act as cues to induce infection-related gene expression. To assign putative function to cDNAs, sequences were initially assembled using StackPack. The estimated total number of genes identified from the three EST databases was 2110: 1088 contigs and 1022 singleton sequences. These 2110 sequences were compared to a yeast protein sequence reference set and to the GenBank nonredundant database using BLASTX. Based on presumptive gene function identified by this process, we found that the two starved cultures had similar, but not identical, patterns of gene expression, whereas the developmental cultures were distinct in their pattern of expression. Of the three libraries, the perithecium library had the greatest percentage (46%) of ESTS falling into the unclassified category. Homologues of some known fungal virulence or pathogenicity factors were found primarily in the N- and C-libraries. Comparisons also were made with ESTs from the related fungi, Neurospora crassa and Magnaporthe grisea and the genomic sequence of N. crassa.
Abstract: Soybean rust is caused by the obligate fungal pathogen Phakopsora pachyrhizi Sydow. A unidirectional cDNA library was constructed using mRNA isolated from germinating P. pachyrhizi urediniospores to identify genes expressed at this physiological stage. Single pass sequence analysis of 908 clones revealed 488 unique expressed sequence tags (ESTs, unigenes) of which 107 appeared as multiple copies. BLASTX analysis identified 189 unigenes with significant similarities (Evalue < 10-5) to sequences deposited in the NCBI non-redundant protein database. A search against the NCBI dbEST using the BLASTN algorithm revealed 32 ESTs with high or moderate similarities to plant and fungal sequences. Using the Expressed Gene Anatomy Classification, 31.7% of these ESTs were involved in primary metabolism, 14.3% in gene/protein expression, 7.4% in cell structure and growth, 6.9% in cell division, 4.8% in cell signaling/cell communication, and 4.8% in cell/organism defense. Approximately 29.6% of the identities were to hypothetical proteins and proteins with unknown function.
Abstract