Using transcription of six Puccinia triticina races to identify the effective secretome during infection of wheat

Abstract

Wheat leaf rust, caused by the basidiomycete Puccinia triticina, can cause yield losses of up to 20% in wheat producing regions. During infection, the fungus forms haustoria that secrete proteins into the plant cell and effect changes in plant transcription, metabolism, and defense. It is hypothesized that new races emerge as a result of overcoming plant resistance via changes in the secreted effector proteins. To understand gene expression during infection and find genetic differences associated with races, RNA from wheat leaves infected with six different rust races, at 6 days post inoculation, was sequenced using Illumina. As P. triticina is an obligate biotroph, RNA from both the host and fungi were present and separated by alignment to the P. triticina genome and a wheat EST reference. A total of 222,571 rust contigs were assembled from 165 million reads. An examination of the resulting contigs revealed 532 predicted secreted proteins among the transcripts. Of these, 456 were found in all races. Fifteen genes were found with amino acid changes, corresponding to putative avirulence effectors potentially recognized by 11 different leaf rust resistance (Lr) genes. Twelve of the potential avirulence effectors have no homology to known genes. One gene had significant similarity to cerato-platanin, a known fungal elicitor, and another showed similarity to fungal tyrosinase, an enzyme involved in melanin synthesis. Temporal expression profiles were developed for these genes by qRT-PCR and show that the genes expression patterns were consistent between races from infection initiation to just prior to spore eruption.

Keywords: Puccinia triticina; RNA sequencing; effectors; leaf rust; secreted peptides.

Figure 1

Infection phenotypes of six P. triticina races on the susceptible spring wheat cultivar Thatcher at six days post inoculation, before sporulating pustules appear. Races are listed at the top. Oil represents the oil only control.

Figure 2

Schematic of bioinformatic pipeline used to identify 15 secreted peptides that are candidates for avirulence effectors.

Figure 3

Gene expression profiles for seven avirulence candidates from P. triticina. Plant samples were taken at 24 h intervals post inoculation (X axis). Real-time PCR was used to quantify expression of each gene. P. triticina Histone H4 (PtHisH4) was used as the internal control for normalization. ΔCq vs. PtHisH4 was used to plot expression (Y axis).