Transcriptome analysis of maize leaf systemic symptom infected by Bipolaris zeicola

Abstract

Bipolaris zeicola is a fungal pathogen that causes Northern corn leaf spot (NCLS), which is a serious foliar disease in maize and one of the most significant pathogens affecting global food security. Here, we report a genome-wide transcriptional profile analysis using next-generation sequencing (NGS) of maize leaf development after inoculation with B. zeicola. We performed High-Throughput Digital Gene Expression analysis to identify differentially expressed genes (DEGs) in resistant inbred Mo17 lines after infection with B. zeicola at four successive disease development stages--CP (contact period), PP (penetration period), IP (incubation period), and DP (disease period); the expression of the genes was compared with those in a CK (mock-treatment) control. In addition, a sensitive maize line (Zheng58) was used for the comparisons with the Mo17. Among all tested genes, 466 differentially expressed genes were identified in all libraries, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of these genes suggested that they are involved in many biological processes related to systemic symptom development, such as plant hormone signal transduction, starch and sucrose metabolism, phenylpropanoid biosynthesis and photosynthesis. Our systematic analysis provides comprehensive transcriptomic information regarding systemic symptom development in fungal-infected plants. This information will help in furthering our understanding of the detailed mechanisms of plant responses to fungal infection.

Fig 1. Defining the stages of maize leaf development in response to B. zeicola and observation of the successive stages of infection by B. zeicola under a scanning electron microscope.

After inoculation, conidia of B. zeicola germinated, and one or two germ tubes were extruded from the poles of the conidia within 2~6 h. Appressorium-like structures were observed in contact with the maize leaf periderm after 12 h, at which time most of the structures had not yet penetrated into leaf surface cells; thus, this time point represents the contact phase (CP) of the disease development. At 24 h, numerous hyphae had differentiated from the germ tubes and were highly branched, and appressoria had directly penetrated the epidermal cell walls, in most cases by developing a constricted penetration peg. However, the fungus also entered through stomata and the intercellular space. This process was considered the penetration period (PP). At 36 h after inoculation, as observed during penetration, the fungus procured host nutrients and grew numerous mycelia on or underneath infected tissues. At the same time, some peridermal cells were collapsed, and a few infected tissues exhibited water-soaked spots, indicating that the infection had developed to the incubation period (IP). After 48h, more water-soaked spots appeared and more obvious symptoms were observed, and this was identified as the end of incubation period—disease periods (DP). The process of disease period was as follows: extensive mycelia colonized on leaf surface and the fungus appeared to have dissolved the cuticle as well as two suberized layers (arrowheads) that indicated mechanical pressure has occurred during host cell wall (HCW) penetration. At last, obvious necrosis appeared on the infected leaf at 96h after inoculation, Meanwhile, a great of new conidia reproduced on necrotic spots, it might be symptoms period (SP)

Fig 2. Determination of SOD, POD, PPO, CAT, and PAL enzyme activity in maize leaves responsive to B. zeicola.

Fig 3. Digital Gene Expression (DGE) analysis of the Mo17 leaf transcriptome responding to B. zeicola infection.

(a) Distribution of reads among the annotated genomic features of maize. (b) Shared and total reads among the phases of symptom development. (c) Distribution of reads among gene models and relative to transcript abundance. (d) Shared and unique reads among the phases of symptom development.

Fig 4. Mapping read information to sense and antisense genes at the five successive stages of symptom development.

Fig 5. Numbers of differently expressed genes among the developmental stages of leaves infected with B. zeicola compared with mock-treatment (Fig. 5a); functional categories and genes grouped according to developmental dynamics using the K-Means clustering algorithm (Fig. 5b).

Fig 6. Cluster and parallel plot of transcription factors expressed at significantly different levels in the five successive stages of infection.

(a) Clusters of transcription factors expressed at significantly different levels in the five successive stages of infection. (b) Parallel plot of transcription factors expressed at significantly different levels in the five successive stages of infection.

Fig 7. Dynamics of transcription factor accumulation profiles.

(a) Dendrogram of transcription factors. Clustering of 527 transcription factors expressed at significantly different levels at the CK phase, during the transition from the CK to the DP phase, and the DP phase clustered into three lineages (G1, G2, and G3) using the self-organization tree algorithm (SOTA). (b) Distribution of transcription factor family proteins among G1, G2 and G3. (c) Distribution of transcription factor families that are expressed at different levels among the CK, CP, IP, PP and DP phases; of which, CP, IP, PP are the transition stage from CK to DP.

Fig 8. GO annotation of DGEs in leaf development in response to B. zeicola infection.

The Y-axis represents the percentage of targeted genes mapped by the GO term and represents the abundance of the GO term. The X-axis expresses the definition of GO terms.