Antifungal activity of rice Pex5p, a receptor for peroxisomal matrix proteins

We have purified a novel antifungal protein from blast fungus (Magnaporthe grisea)-treated rice leaves using consecutive chromatographies on CM-Sepharose ion-change, Affi-gel blue, and HPLC gel filtration columns. We determined the N-terminal peptide sequence of the purified protein and subjected it to the NCBI/BLAST database and found the protein to be a partial fragment of the peroxisomal receptor protein in rice (OsPex5p). After cloning two cDNAs encoding OsPEX5L and OsPEX5S genes that are splice variants of OsPEX5 from a rice leaf cDNA library, we investigated their antifungal properties. The recombinant proteins were expressed in Escherichia coli and found to significantly inhibit cell growth of various pathogenic fungal strains. mRNA expression of the OsPEX5L gene was induced by diverse external stresses such as rice blast fungus, fungal elicitor, and other signaling molecules including H(2)O(2), abscisic acid, jasmonic acid, and salicylic acid. These results suggest that the peroxisomal receptor protein, OsPex5p, plays a critical role in the rice defense system against diverse external stresses including fungal pathogenic attack.     

Induction of thaumatin‐like proteins (TLPs) in Rhizoctonia solani‐infected rice and characterization of two new cDNA clones

Thaumatin‐like proteins (TLPs) were shown to be induced in rice plants (cv. IR58) that were infected with the sheath blight fungus, Rhizoctonia solani . Western blot analysis revealed the presence of two TLPs with sizes of 25 and 24 kDa which are different from a previously reported TLP with a size of 15.6 kDa from rice plants infiltrated with the non‐pathogenic bacterium, Pseudomonas syringae pv. syringae . By probing a cDNA expression library prepared from RNA isolated from R. solani ‐infected rice plants with a TLP antibody, several putative TLP cDNA clones were isolated and sequenced. The cDNA clones appeared to be derived from two different genes which shared only 77% sequence identity with each other and a lower percentage of sequence identity with the previously reported TLP cDNA clone. Southern blot analysis with the two TLP cDNAs revealed different rice genomic DNA fragments. Northern blot analysis also confirmed that a 1.1‐kb RNA detectable by the TLP cDNA inserts was induced by fungal infection. Thus rice TLPs are encoded by a family of at least three genes which are differentially expressed in responses to bacterial or fungal pathogens.     

Analysis of genes expressed during rice-Magnaporthe grisea interactions.

Expressed sequence tag (EST) analysis was applied to identify rice genes involved in defense responses against infection by the blast fungus Magnaporthe grisea and fungal genes involved in growth within the host during a compatible interaction. A total of 511 clones was sequenced from a cDNA library constructed from rice leaves (Oryza sativa cv. Nipponbare) infected with M. grisea strain 70-15 to generate 296 nonredundant ESTs. The sequences of 293 clones (57.3%) significantly matched National Center for Biotechnology Information database entries; 221 showed homologies with previously identified plant genes and 72 with fungal genes. Among the genes with assigned functions, 32.8% were associated with metabolism, 29.4% with cell/organism defense or pathogenicity, and 18.4% with gene/protein expression. cDNAs encoding a type I metallothionein (MTs-1) of rice and a homolog of glucose-repressible gene 1 (GRG1) of Neurospora crassa were the most abundant representatives of plant and fungal genes, comprising 2.9 and 1.6% of the total clones, respectively. The expression patterns of 10 ESTs, five each from rice and M. grisea, were analyzed. Five defense-related genes in rice, including four pathogenesis-related genes and MTs-1, were highly expressed during M. grisea infection. Expression of five stress-inducible or pathogenicity-related genes of the fungus, including two hydrophobin genes, was also induced during growth within the host. Further characterization of the genes represented in this study would be an aid in unraveling the mechanisms of pathogenicity of M. grisea and the defense responses of rice.     

A rice isoflavone reductase-like gene, OsIRL, is induced by rice blast fungal elicitor

We have isolated and characterized a rice isoflavone reductase-like gene, OsIRL, whose expression is induced by a fungal elicitor. The OsIRL cDNA contains 1203 bp with an open reading frame of 942 nucleotides encoding 314 amino acids. The deduced amino acid sequence of OsIRL has a putative pyridine nucleotide binding domain and is 68% homologous with the maize isoflavone reductase-like gene. Southern blot analysis revealed that OsIRL belongs to a small multigene family. Expression of OsIRL was induced by treatment with a fungal elicitor and jasmonic acid as well as by inoculation with rice blast fungus. Cycloheximide (1 microM), strongly inhibited the induction of OsIRL by the fungal elicitor, indicating that new protein synthesis is required. The protein kinase inhibitor, staurosporine (1 microM), had little effect, but the phosphatase inhibitor, calyculin A (1 microM), strongly inhibited induction. Treatment with salicylic acid (SA, 5 mM) strongly inhibited expression of OsIRL in response to fungal elicitor and JA, while abscisic acid (ABA, 200 microM) also strongly antagonized OsIRL induction by JA, but had only a weak effect on induction by the fungal elicitor. These results suggest that the expression of OsIRL is positively regulated by phytohormones such as JA, and negatively by phytohormones such as SA, ABA.     

Identification of rice genes induced in a rice blast-resistant mutant

To clarify mechanisms of rice blast resistance in rice plants we used suppression subtractive hybridization (SSH) to isolate genes induced upon rice blast inoculation in a rice blast-resistant mutant. A total of 26 rice cDNAs were isolated and found to have elevated expression upon rice blast infection in a rice blast-resistant derivative, SHM-11, of the rice cultivar, Sanghaehyanghyella. Sequencing of the cDNAs revealed that many of the proteins they encoded had been previously described as involved in plant responses against pathogen attack. Two interesting groups of the defense-related proteins consisted of three different PR5 homologues and four different protease inhibitors, all highly expressed in the rice blast mutant. Genes encoding proteins involved in signal transduction and regulation were also identified, including translation initiation factor eIF5A, C2 domain DNA binding protein, putative rice EDS and putative receptor like kinase. Most of the identified cDNAs were highly expressed 24 h after blast inoculation. Our results suggest that a pathway regulating defense gene expression may be altered in the mutant, resulting in early induction of the defense genes upon fungal infection.     

Comparative study of genes expressed from rice fungus-resistant and susceptible lines during interactions with Magnaporthe oryzae

Rice blast disease caused by Magnaporthe oryzae is the most important fungal disease of rice. To understand the molecular basis of interaction between the fungus and rice, we constructed a cDNA library from a rice-resistant line inoculated with M. oryzae. One hundred and fifty-three cDNA clones were sequence analyzed, of which 129 exhibited significant nucleotide sequence homology to known genes, 21 were homologous to unknown genes, while three clones did not match to any database. However, these three unmatched clones showed sequence homology at protein level in the protein databases and one of them encoded a disease resistance-related protein kinase and was abundant in the EST collection. Northern analysis showed that this disease resistance-related protein kinase gene was induced by inoculation and only expressed in the rice-resistant, but not susceptible, lines. Southern analysis showed that this gene was present in a single copy in the rice genome and co-segregated with the M. oryzae resistance in the cross of the resistant and susceptible lines. This study illustrates that sequencing of ESTs from inoculated resistant plants can reveal genes responsive to pathogen infection, which could help understand plant defense mechanisms.     

Rice small C2-domain proteins are phosphorylated by calcium-dependent protein kinase

We previously reported that OsERG1 and OsERG3 encode rice small C2-domain proteins with different biochemical properties in Ca²⁺- and phospholipid-binding assays. Os-ERG1 exhibited Ca²⁺-dependent phospholipid binding, which was not observed with OsERG3. In the present study, we show that both OsERG1 and OsERG3 proteins exhibit oligomerization properties as determined by native polyacrylamide gel electrophoresis (PAGE) and glutaraldehyde cross-linking experiments. Furthermore, in vitro phosphorylation assays reveal the phosphorylation of OsERG1 and OsERG3 by a rice calcium-dependent protein kinase, OsCDPK5. Our mutation analysis on putative serine phosphorylation sites shows that the first serine (Ser) at position 41 of OsERG1 may be an essential residue for phosphorylation by OsCDPK5. Mutation of Ser41 to alanine (OsERG1S41A) and aspartate (OsERG1S41D) abolishes the ability of OsERG1 to bind phospholipids regardless of the presence or absence of Ca²⁺ ions. In addition, unlike the OsERG1 wild-type form, the mutant OsERG1 (S41A)::smGFP construct lost the ability to translocate from the cytosol to the plasma membrane in response to calcium ions or fungal elicitor. These results indicate that Ser41 may be essential for the function of OsERG1.