CLONING AND CHARACTERIZATION OF PWL1 AND PWL2 HOST-SPECIES SPECIFICITY GENES OF THE FINGER MILLET BLAST PATHOGEN, MAGNAPORTHE ORYZAE

Abstract

Blast disease, caused by the filamentous ascomycete fungus, Magnaporthe oryzae, is the most devastating disease affecting finger millet (Eleusine coracana subsp. coracana) production in eastern Africa. The fungus also infects many other grass species in the poaceae family. It produces effector molecules to manipulate host immunity and metabolic processes as they successfully invade live host, and in this case, finger millet cells. Some of these effectors have avirulence (AVR) activities and therefore are recognized by the host plant’s R genes, preventing completion of the infection cycle since blast infection mainly depends on the interaction between the pathogen’s effector genes and the host’s resistant (R) proteins. This study investigated the distribution of three members of a rapidly evolving gene family, the PWL genes, in 226 finger millet blast isolates collected from eastern Africa, to examine the evolution of these effector genes. This was further narrowed down to understand the roles PWL1 and PWL2 play in the finger millet blast pathogen towards pathogenicity on weeping lovegrass (Eragrostis curvula) and other host plants. PWL1 and PWL2 were amplified from a finger millet blast isolate E22, using high fidelity PCR. It was cloned and transformed individually into U34, a finger millet blast isolate lacking both PWL1 and PWL2, using Agrobacterium tumefaciens mediated transformation and followed by infection assays on a panel of grasses. Results from this study showed that transformed isolate U34 lost its pathogenicity on weeping lovegrass but remained fully pathogenic on finger millet. Contrary to the current understanding of infections involving AVR genes, it was established for the first time, that presence of PWL1 and PWL2 prevented the fungus from infecting the grass host Sporobolus phyllotrichus. The study further revealed that the 226 finger millet blast isolates studied, grouped into two distinct classes, differentiated by the presence or absence of PWL1 and PWL2 and their pathogenicity towards weeping lovegrass and Sporobolus phyllotrichus. EC-1 isolates, did not have PWL1 or PWL2 and were highly virulent on both finger millet, weeping lovegrass and Sporobolus phyllotrichus. EC-2 isolates contained either PWL1 or PWL2 or both and did not infect weeping lovegrass and Sporobolus phyllotrichus but were highly virulent on finger millet. The avirulence of EC-2vi isolates on weeping lovegrass and Sporobolus phyllotrichus seemed to be conditioned by the presence of PWL1 and/or PWL2. Results further indicated that all finger millet blast isolates with PWL4 were pathogenic on weeping lovegrass and Sporobolous phyllotrichus. It therefore suggested that PWL4 does not modulate the infectivity of the pathogen. Gene copy number variations, shared non-synonymous mutations and high levels of nucleotide variation diversity were observed. Therefore, non-synonymous mutations and/or loss and gain of AVR genes might be the main evolutionally mechanism underlying the rapid evolution of this fungus in relation to virulence towards the host plant. Findings from this study improved the understanding of the function and potential mechanism underlying the evolution and dynamics of the PWL gene family.