Identification And Functional Assessment Of Early Response Genes Of Wheat Induced By Powdery Mildew

Powdery mildew, caused by Blumeria graminis f.sp. tritici, is one of the most diseases in wheat(Triticum aestivum L.) worldwide. In wheat, race-specific resistance to the fungal pathogen powderymildew is controlled by Pm genes. This research aims at: 1) revealing the characteristics of Pro-mediatedresistance response at the cytological level; 2) screening and functional assessing early response genes ofwheat that are induced by powdery mildew in an attempt to give a rough explanation for the mediatingmechanism of Pm genes. By use of primary leaves of three susceptible lines (Bainong3217, Beijing 837and Jingshuang16) and five resistant lines (Mardler (Pm2+Pm6), Ulka/8*Cc (Pm2),Mardler/7*Bainong3217 (Pm2), Pm16 and Pm16/7*Beijing837 (Pm16)), the early hydrogen peroxide(H₂O₂) accumulations of compatible and incompatible interactions from 10 to 48 hours after inoculation(hai) were compared. Early expressed genes that might be differentially regulated following inoculation ofpowdery mildew were identified by comparison of resistant (Bainong3217-Pm2-line) and susceptible(Bainong3217) wheat with the powdery mildew fungus via cDNA-AFLR Comparative analysis of theLTP1 coding regions in different genome materials was carried out. Expression patterns of LTP genes(LTP1+LTP2) upon different stresses were investigated. Functions of these early expressed candidate genesand LTP1 were further assessed by transient assay system. The results obtained are as follows:1. I compared the H₂O₂ accumulation condition in wheat leaf cells in response to powdery mildewprimary germ tube (PGT) contact, appressorial germ tube (AGT) contact, in papilla (cell wall apposition)and HR in wheat. The results showed that: 1) H₂O₂ is important in effective papilla formed as a generalplant defense to powdery mildew; 2) five resistant lines had greater frequency of effective papilla formationcompared with the susceptible lines; 3) among resistant wheat lines, hypersensitive cell death (HR) inMardler and Ulka/8*Cc was detected earlier than in Pm16 and Pm16/7*Beijing837. In all cases HR wasassociated with H₂O₂ accumulation throughout attacked epidermal cells; 4) interestingly, penetrationresistance, not HR, appeared to be mediated by the Pro2 gene in the Mardler/7*Bainong3217 line,suggesting that this gene may govern an HR-independent defense pathway in this genetic background; 5) inwheat-powdery mildew incompatible interaction, HR may act as a second line of defense to containinfection when the papilla defense fails.2. Differential gene expression of wheat after powdery mildew inoculation was studied bycDNA-AFLR A total of 10 000 cDNA fragments were screened. Two hundred and eighty three (2.8ï¼…)showed differential expression 1, 3, 6, 12 and/or 24 hours after inoculation (hai). Of these transcript derivedfragments (TDFs), 61 were sequenced and inductions by pathogens were further confirmed via RT-PCR in3 cDNA fragments out of the 7 tested. Two TDFs were found to be homologous to a CDPK (TaCDPK2)and aperoxiredoxin Q (TaPrxl) and their full length cDNAs were isolated. TaCDPK2 (acc. No. AY704444)codes for a protein with 558 amino acids. The typical domains found in all known CDPKs are present inthe deduced protein: A kinase domain, consistent with the structure of serine/threonine protein kinases(amino acids 96-360), is connected to a cahnodulin-like domain (amino acids 392-535) by a short junctiondomain (31 amino acids) and the cahnodulin-like region contains four canonical EF-hand Ca²⁺-bindingmotifs. Therefore, TaCDPK2 is not only involved in salt/draught tolerance but also in fungal defense. Thefunction of TaCDPK indicated that it might be a general fungal defense gene rather than a specific gene inresponse to powdery mildew of resistant plants. TaPrxl (acc. No. AY789643) codes for a protein with 217amino acids. The typical domain found in known prx Q of Arabidopsis is present in the deduced protein:alkyl hydroperoxide reductase (AhpC) and thiol specific antioxidant (TSA) (amino acids 73-196). Theup-regulated expression of TaPrxl was 3 hours earlier in Mardler/7*Bainong3217 (resistant) than in Bainong3217 (susceptible) after powdery mildew inoculation. Over expression of TaPrxl via biolisticdelivery of full-length cDNA to wheat leaf showed that it reduced the penetration efficiency (PE) ofpowdery mildew on susceptible cultivar more pronounced than on the control (pGY1 empty vector). Thissuggests that the peroxiredoxin Q of wheat (TaPrxl) is a candidate gene for the early regulator of powderymildew resistance. Expression analysis also showed that TaPrxl may be generally involved inenvironmental (cold/salt/draught) stress response. The third TDF A27 was 86% identical on the amino acidlevel to an auxin response factor 2 from rice. A 27 was isolated as a 608 bp cDNA fragment containing B3and auxin_response conserved domains. Only in compatible wheat-powdery mildew interaction, theexpression of A 27 was activated at early stage (1, 3, 6 hai) after powdery mildew inoculation. Thissuggested that ARF2 of wheat could be involved in susceptible response to fungal pathogen infection. Thisis the first report on the possible cross-talk between host fungal responses and auxin response pathways.3. Wheat LTP1 gene is involved in powdery mildew resistance; LTP genes of wheat can be induced byboth salt and PEG stresses and expressed in different levels in different tissues; LTP1 and LTP2 areconservative in evolution; LTP4 from biploid UR102 (A) is a new type of LTP.