Serotonin attenuates biotic stress and leads to lesion browning caused by a hypersensitive response to Magnaporthe oryzae penetration in rice.

The hypersensitive response (HR) of plants is one of the earliest responses to prevent pathogen invasion. A brown dot lesion on a leaf is visual evidence of the HR against the blast fungus Magnaporthe oryzae in rice, but tracking the browning process has been difficult. In this study, we induced the HR in rice cultivars harboring the blast resistance gene Pit by inoculation of an incompatible M. oryzae strain, which generated a unique resistance lesion with a brown ring (halo) around the brown fungal penetration site. Inoculation analysis using a plant harboring Pit but lacking an enzyme that catalyzes tryptamine to serotonin showed that high accumulation of the oxidized form of serotonin was the cause of the browning at the halo and penetration site. Our analysis of the halo browning process in the rice leaf revealed that abscisic acid enhanced biosynthesis of serotonin under light conditions, and serotonin changed to the oxidized form via hydrogen peroxide produced by light. The dramatic increase in serotonin, which has a high antioxidant activity, suppressed leaf damage outside the halo, blocked expansion of the browning area and attenuated inhibition of plant growth. These results suggest that serotonin helps to reduce biotic stress in the plant by acting as a scavenger of oxygen radicals to protect uninfected tissues from oxidative damage caused by the HR. The deposition of its oxide at the HR lesion is observed as lesion browning.

Effects of Pyramiding Quantitative Resistance Genes pi21, Pi34, and Pi35 on Rice Leaf Blast Disease.

Development of resistant cultivars has been an effective method for controlling rice blast disease caused by Magnaporthe oryzae. Quantitative blast resistance genes may offer durable resistance because the selection pressure on M. oryzae to overcome resistance is low as a result of the genes' moderate susceptibility. Because the effects of individual resistance genes are relatively small, pyramiding these genes in rice cultivars is a promising strategy. Here, we used near-isogenic and backcross lines of rice cultivar Koshihikari with single- or two-gene combinations of blast resistance genes (pi21, Pi34, and Pi35) to evaluate the suppression of leaf blast. The severity of the disease was assessed throughout the infection process. Resistance varied among the lines: Pi35 conferred the strongest resistance, while Pi34 showed the weakest effects. Two types of combined-gene interactions were observed, and they varied on the basis of gene combination and characteristic of the infection: (i) the combination of two resistance genes was more effective than either of the genes individually or (ii) the combination of two resistance genes was similar to the level of the most effective resistance gene in the pair. The most effective gene combination for the suppression of leaf blast was pi21 + Pi35.

Loss of a 1.6 Mb chromosome in Pyricularia oryzae harboring two alleles of AvrPik leads to acquisition of virulence to rice cultivars containing resistance alleles at the Pik locus.

A small and extra chromosome of 1.6 Mb was previously identified in a Pyricularia oryzae strain, 84R-62B. To understand a role of the 1.6 Mb chromosome in the pathogenic changeability of P. oryzae, we performed experiments designed to characterize the 1.6 Mb chromosome in the present study. A gene family encoding secreted protein Pex31s in P. oryzae consists of five homologs, Pex31-A to -E. Among them, Pex31-A and -D are known to be recognized by Pik-m and Pik/Pik-m/Pik-p, respectively. In the present study, we identified Pex31-A and -D in the genome of 84R-62B. Segregation analyses using an F1 population between 84R-62B and another rice blast strain, Y93-245c-2, revealed a strong linkage between the two homologs and the 1.6 Mb chromosome of 84R-62B. A CHEF-Southern analysis revealed an association between the 1.6 Mb chromosome and the homologs, indicating that both homologs are located on the 1.6 Mb chromosome of 84R-62B. The loss of the 1.6 Mb chromosome was observed in subcultures of a F1 progeny, F1-327. These subcultures concomitantly acquired virulence on Pik, Pik-m, and Pik-p. The present study is the first report showing that loss of a small and extra chromosome leads to pathogenic mutation of P. oryzae and may provide a new insight into the mechanisms generating pathogenic variation of this fungus.

Multiple translocation of the AVR-Pita effector gene among chromosomes of the rice blast fungus Magnaporthe oryzae and related species.

Magnaporthe oryzae is the causal agent of rice blast disease, a devastating problem worldwide. This fungus has caused breakdown of resistance conferred by newly developed commercial cultivars. To address how the rice blast fungus adapts itself to new resistance genes so quickly, we examined chromosomal locations of AVR-Pita, a subtelomeric gene family corresponding to the Pita resistance gene, in various isolates of M. oryzae (including wheat and millet pathogens) and its related species. We found that AVR-Pita (AVR-Pita1 and AVR-Pita2) is highly variable in its genome location, occurring in chromosomes 1, 3, 4, 5, 6, 7, and supernumerary chromosomes, particularly in rice-infecting isolates. When expressed in M. oryzae, most of the AVR-Pita homologs could elicit Pita-mediated resistance, even those from non-rice isolates. AVR-Pita was flanked by a retrotransposon, which presumably contributed to its multiple translocation across the genome. On the other hand, family member AVR-Pita3, which lacks avirulence activity, was stably located on chromosome 7 in a vast majority of isolates. These results suggest that the diversification in genome location of AVR-Pita in the rice isolates is a consequence of recognition by Pita in rice. We propose a model that the multiple translocation of AVR-Pita may be associated with its frequent loss and recovery mediated by its transfer among individuals in asexual populations. This model implies that the high mobility of AVR-Pita is a key mechanism accounting for the rapid adaptation toward Pita. Dynamic adaptation of some fungal plant pathogens may be achieved by deletion and recovery of avirulence genes using a population as a unit of adaptation.

Molecular cloning and characterization of the AVR-Pia locus from a Japanese field isolate of Magnaporthe oryzae.

In order to clone and analyse the avirulence gene AVR-Pia from Japanese field isolates of Magnaporthe oryzae, a mutant of the M. oryzae strain Ina168 was isolated. This mutant, which was named Ina168m95-1, gained virulence towards the rice cultivar Aichi-asahi, which contains the resistance gene Pia. A DNA fragment (named PM01) that was deleted in the mutant and that co-segregated with avirulence towards Aichi-asahi was isolated. Three cosmid clones that included the regions that flanked PM01 were isolated from a genomic DNA library. One of these clones (46F3) complemented the mutant phenotype, which indicated clearly that this clone contained the avirulence gene AVR-Pia. Clone 46F3 contained insertions of transposable elements. The 46F3 insert was divided into fragments I-VI, and these were cloned individually into a hygromycin-resistant vector for the transformation of the mutant Ina168m95-1. An inoculation assay of the transformants revealed that fragment V (3.5 kb) contained AVR-Pia. By deletion analysis of fragment V, AVR-Pia was localized to an 1199-bp DNA fragment, which included a 255-bp open reading frame with weak homology to a bacterial cytochrome-c-like protein. Restriction fragment length polymorphism analysis of this region revealed that this DNA sequence co-segregated with the AVR-Pia locus in a genetic map that was constructed using Chinese isolates.