Necrosis-Inducing Proteins ofRhynchosporium commune, Effectors in Quantitative Disease Resistance

Abstract

The barley pathogen Rhynchosporium commune secretes necrosis-inducing proteins NIP1, NIP2, and NIP3. Expression analysis revealed that NIP1 transcripts appear to be present in fungal spores already, whereas NIP2 and NIP3 are synthesized after inoculation of host plants. To assess the contribution of the three effector proteins to disease development, deletion mutants were generated. The development of these fungal mutants on four barley cultivars was quantified in comparison with that of the parent wild-type strain and with two fungal strains failing to secrete an "active" NIP1 avirulence protein, using quantitative polymerase chain reaction as well as microscopic imaging after fungal green fluorescent protein tagging. The impact of the three deletions varied quantitatively depending on the host genotype, suggesting that the activities of the fungal effectors add up to produce stronger growth patterns and symptom development. Alternatively, recognition events of differing intensities may be converted into defense gene expression in a quantitative manner.

PFP1 , a Gene Encoding an Epc-N Domain-Containing Protein, Is Essential for Pathogenicity of the Barley Pathogen Rhynchosporium commune

“…Successful deletion was confirmed by a third PCR analysis, in which the 5= and 3= integration sites were amplified (primers 5=-Int1s/ 5=-Int3s and 5=-Int2as and primers 3=-Int1s/3=-Int3s and 3=-Int4as), and by Southern hybridization. Before further characterization, fungal mutants were grown on barley cultivar 'Ingrid' and reisolated (13).…”

“…By application of the efficiency calibrated model (42), the GPD gene (primers GPD-RTs and GPD-RTas) served as the fungal target gene and a gene encoding a putative member of the high-temperature-requirement A (HTRA) family of serine proteases (GenBank accession no. AK359241; primers HO05J24F and HO05J24R) served as the barley reference gene (13). To quantify fungal growth in liquid culture, fungal cultures were initiated with 10 3 or 10 4 spores in sucrose-supplemented Fries medium no.…”

“…To quantify fungal gene expression, total RNA was extracted from inoculated barley leaves or from fungal mycelia grown ex planta either manually (mycelia were ground with a mortar and pestle) or using a cryogenic grinder (Labman Automation Ltd., Stokesley, North Yorkshire, United Kingdom). Transcripts of the PFP1 target gene (primers PFP-RTs and PFP-RTas) were quantified relative to those of the constitutively expressed GPD gene (primers GPDRTs and GPD-RTas) by quantitative reverse transcription-PCR (qRT-PCR) (13,42). The absence of genomic DNA was routinely tested by amplifying the GPD gene sequence.…”

“…The typical disease symptoms, necrotic lesions, occur after a latent period lasting from a few days up to several weeks, when mesophyll cells in leaf regions that are heavily colonized by the fungus collapse (12,13). Hence, fungal growth in planta can be divided into 4 phases: germination and penetration, early development of thin hyphae with a very slow gain of fungal biomass, exponential growth of mainly thick hyphae with a massive increase in biomass, and late stationary phase with sporulation (13).…”

“…No biochemical activity has been able to be attributed to NIP2 to date, whereas NIP1 and NIP3 have been found to stimulate the plant plasma membrane-localized H ϩ -ATPase (16). The three NIP genes show strong expression during the early fungal growth stage, followed by a decline during the period of rapid fungal growth, and they contribute quantitatively to fungal virulence (13). Furthermore, NIP1 was shown to be a dual-function effector (17): on top of its nonspecific virulence activity, the protein is the avirulence factor corresponding to barley resistance gene Rrs1 (18,19).…”