Identification And Functional Analysis Of Proteins Associated With Resistance To Curvularia Leaf Spot Of Maize

Maize leaf spot caused by Curvularia lunata (Wakker) Boed is one of the most widely distributed diseases in maize growing area of China. The disease ever outbroke seriously and resulted in a great grain loss in 1996 in northern China. In the long run, breeding resistant cultivars is the most economic and effective measures to control this disease. However, until now few cultivars possess high resistance to the disease. Furthermore, the resistance of cultivars may be easily lost due to the frequent genetic variance of the pathogen. Thus exploring proteins or genes related to the host resistance from tropic germplasm appears to be the most pressing task. Here, a proteomic approach mainly including 2-dimensional gel electrophoresis (2-DE) and MALDI-TOF MS/MS techniques was applied to screen and identify defense-related proteins from resistant inbred lines widely planted in China, then genes encoding corresponding proteins were cloned, eventually their functions in host resistance were discussed. The main results were as follows:1. Changes of defense-related factors in maize response to C. lunata infection. After inoculation with C. lunata, the content of SA and ROS especially O2- was significantly increased in resistant inbred lines. Besides, the transcription levels of defense genes including pathogenesis-related genes PR-1, PR-10, PAL, CAT, and POX were also significantly enhanced. While the content of ABA was found markedly increased in susceptible ones, and the transcription levels of defense genes were also increased to some extent but not significantly. Based on the results, SA was speculated to be positively correlated with maize resistance to C. lunata attack, but ABA was on the contrary. The accumulation of ROS in the early stage of pathogen infection was detected and inferred to stimulate the enhancement of maize disease resistance. Meanwhile PR-1, PR-10, PAL and ROS scavenging enzyme also played important roles in maize resistance to C. lunata infection.2. Optimized 2-DE system to separate proteins from maize leaves. A 2-DE system to effectively extract and separate total maize leaf proteins was established by optimizing protein extracting methods, sample lysis buffer, pH value of IPG gel and time for IEF. Meanwhile, Mg/NP-40 with PEG fraction was evidenced to be an effective method to remove high abundant proteins mainly Rubisco from total leaf protein samples according to Kim's methods (2001). The capacity of 2-DE system to visualize and detect low abundant proteins improved greatly. 3. Identification of proteins associated with resistance to Curvularia leaf spot of maize. After inoculation with C. lunata, proteomic profiles of maize leaves between resistant and susceptible inbred lines were analyzed. Thirty-six differentially expressed protein spots resolved on the 2-DE gels were successfully identified by MALDI-TOF MS/MS. The results showed that the proteins were associated with photosynthesis, respiration, drought and oxidative stress tolerance as well as signal transduction. Of those stress related proteins, 22kDa drought-inducible protein, ABA/stress/ripening inducible protein (Asr), germin-like protein (GLP), glutathione peroxidase (GPX), ascorbate peroxidase (APX), translation initiation factor (eIF-5A) and ras related protein were unique or up-regulated in the resistant inbred lines and supposed to be implicated in host defense response to C. lunata infection. Meanwhile, drought and oxidative stress-related proteins were primarily considered to be most closely involved in maize resistance to pathogen infection. Based on the above and other observations, it was deduced that there might be cross-talk between host resistance to abiotic and biotic stresses.4. Cloning of defense-related gene ZmDip and its expression. Accoding to mass spectrometry result,the gene encoding 22 kDa drought inducible protein in maize was successfully cloned through RT-PCR and named as ZmDip. To carry out gene function analysis, the high level expression of ZmDip in E. coli was achieved, and subsequently purified for antibody preparation. Besides, ZmDip (Genbank accession No. EU164846) showed 98% and 99% consistency in gene and protein sequence with that of ZmAsr1 ( AX297905 , NCBI ) , respectively, so they were supposed to be one gene even though the minor difference existed, which was perhaps partly attributed to genotypes of maize inbred lines.5. Analysis of transcription levels of ZmDip in normal and inoculated conditions. The expression patterns of ZmDip in different tissues of maize were analyzed with semi-quantitative RT-PCR. The gene was constitutively expressed in root but not in leaf sheath. Its expression was very complicated in leaves, and depended probably on genotypes of maize inbred lines. Transient expression of ZmDip in maize leaves was induced when treated with ABA and SA. Moreover, differential transcript levels of ZmDip was found in resistant or susceptible inbred lines after inoculated with C. lunata, but no clear trend was found regarding to their correlation with maize resistance levels and content of ABA and SA in inoculated host. Thus it was deduced that ZmDip might be mainly regulated by other singal pathways besides ABA and SA.6. Expression of ZmDip improved host resistance to C. lunata infection. To discover the roles of ZmDip played in maize resistance to C. lunata infection, the recombinant vector pBI-ZmDip-Gus was constructed. ZmDip successfully expressed in leaves in vitro (Huangzao 4) through Agrobacterium-mediated transformation. The expression of ZmDip led to the accumulation of ROS specially O2- and remarkably reduced leaf spot area in transformed leaf tissue by subsequent challenge inoculation as compared with control. Taking together, ZmDip played a positive role in strengthening maize resistance to C. lunata infection, but the exact role ZmDip played needed to be further discussed.